CN109706155B - OsHEN1 OsSPL14 gene expression box and construction method and application thereof - Google Patents

Abstract

The invention provides a pOsHEN1 gene expression cassette OsSPL14 gene, a pOsHEN1 gene expression cassette OsSPL14 gene comprising an OsHEN1 promoter and an OsSPL14 gene, wherein the OsSPL14 gene is connected with the downstream of the OsHEN1 promoter, and the invention also provides a recombinant expression vector containing the pOsHEN1 gene expression cassette OsSPL14 gene expression cassette and a construction method of the recombinant expression vector. The gene expression cassette and the recombinant expression vector can generate rice excellent yield traits such as increased stem thickness, increased tiller yield and/or increased resistance of rice to rice bacterial blight PXO 99A. When the transgenic strain line is not invaded by PXO99A, the expression quantity of the OsSPL14 of the background in the transgenic rice is slightly increased, the characteristic that the OsSPL14 regulates the ideal plant type of the rice is exerted, the tillering is properly reduced to improve the effective tillering number, the stem thickness lodging resistance is increased, and the rice tillering yield is improved. When a transgenic line is invaded by PXO99A, the transcription level of the downstream OsSPL14 gene is greatly improved, and the disease resistance to the bacterial blight can be specifically enhanced.

Description

OsHEN1 OsSPL14 gene expression box and construction method and application thereof

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to construction and breeding application of a pOsHEN1 gene expression cassette OsSPL 14.

Background

Xanthomonas oryzae pv. oryzae, Xoo is a pathogenic bacterium causing bacterial blight of rice. The bacterial leaf blight of rice occurs in each rice area in China, and is a main disease of rice. The rice seedlings have short and narrow scabs at high temperature, the expanded leaves quickly wither and wither, the number of blighted grains and broken rice is large, the yield is reduced by 20 to 30 percent, the weight can reach 50 to 60 percent, even the grains are not harvested, and the influence on the yield is large. Therefore, it is highly necessary to improve the bacterial blight resistance of rice by genetic engineering.

Plants, including crops, are mostly fixed in growing areas, and can change the signal network of endogenous growth and development according to the changing external environment, so that the plants are better adapted to the external environment. When infected by pathogenic bacteria, plants regulate energy from the aspect of growth and development to defense signal networks, so that the process of resisting diseases is often expressed as growth and development inhibition. In order to improve disease resistance of crops, relevant research in laboratories screened a group of mutants that constitutively activated resistance responses. The mutants are usually shown as cell death, high expression of disease-resistant related genes and high accumulation of resistant substances, and can finally effectively inhibit the propagation of bacteria or fungi, but the mutants have the defects of plant dwarfing, reduced fertility, reduced seed yield and the like in the aspects of growth and development. The study of these disease-resistant genes has contributed greatly to the understanding of plant immune molecular pathways, but the use of these genes in genetic breeding production has been hampered because of the often concomitant developmental defects mentioned above.

Therefore, in the research of improving the bacterial blight resistance of rice, the opposite aspects of stress resistance and development of rice need to be balanced. The research of the transgenic material which can not only increase the disease resistance of the rice specifically, but also increase the yield of the rice has important and wide application value.

Disclosure of Invention

The invention aims to provide a gene expression cassette which can specifically increase the bacterial leaf blight resistance of rice and can also increase the rice yield, a construction method and application thereof aiming at the defects of the prior art.

The technical scheme of the invention is as follows:

the first object of the invention is to provide a pOsHEN1 OsSPL14 gene expression cassette, the pOsHEN1 OsSPL14 gene expression cassette comprises an OsHEN1 promoter and an OsSPL14 gene, and the OsSPL14 gene is connected with the downstream of the OsHEN1 promoter.

The nucleotide sequence of the OsHEN1 promoter is shown as SEQ ID No.5, and the nucleotide sequence of the OsSPL14 gene is shown as SEQ ID No. 6.

The second purpose of the invention is to provide a recombinant expression vector containing the OsHEN1 gene expression cassette OsSPL 14.

Furthermore, the recombinant expression vector is obtained by inserting the pOsHEN1:: OsSPL14 gene expression cassette of claim 1 between EcoRI and HindIII sites of pCAMBIA1305.1 plasmid vector.

The application of the OsSPL14 gene expression cassette or the recombinant expression vector in improving rice crops comprises the following steps: improving the excellent yield traits of rice and/or improving the resistance of the rice to rice bacterial blight strain PXO 99A.

Further, the rice superior yield traits include: reducing tillering to increase effective tillering number, reducing production consumption, increasing stem thickness, enhancing lodging resistance, and/or increasing single ear yield of rice to increase rice yield

The third object of the present invention is to provide the construction method of the aforementioned recombinant expression vector, which comprises the following steps:

s1: taking rice Nipponbare DNA as a template, and taking OsHEN1 promoter specific primers pOsHEN 1-F:

tatgaccatgattacgaattcTTATGTGCACTAGAAACTATCTGAGGAC (SEQ ID No.1) and pOsHEN 1-R: CAAACGCCCAAAAAAAACAA (SEQ ID No.2) to carry out PCR amplification, and cloning out a promoter amplification product of OsHEN 1;

s2: taking rice Nipponbare DNA as a template, and taking an OsSPL14 gene specific primer OsSPL 14-F:

ttgttttttttgggcgtttgTTCCGTCTCTTTCCTCTCTCTTCT (SEQ ID No.3) and OsSPL 14-R:

tggtctttgtagtcaagcttCAGAGACCAATCCATCGTGTTG (SEQ ID No.4), and cloning a genomic DNA amplification product of OsSPL 14;

s3: the amplified fragments obtained from S1 and S2 were ligated to EcoRI and HindIII double digested plasmid vector pCAMBIA1305.1 by homologous recombination to obtain a recombinant vector containing the expression cassette OsHEN1: OsSPL 14.

Further, the specific operation steps of S3 are: a homologous recombination kit with the product number of C113-02 of Nanjing Novozan company is utilized to connect a promoter fragment of OsHEN1, a genomic DNA fragment of OsSPL14 and a pCAMBIA1305.1 linear fragment subjected to double enzyme digestion of EcoRI and HindIII together, and a recombinant vector containing a pOsHEN1: OsSPL14 expression cassette is obtained through Escherichia coli transformation and monoclonal identification.

The homologous recombination system is as follows:

and (3) configuring a recombinant system according to the system, sucking and beating the recombinant system by a pipette gun, uniformly mixing the recombinant system and the pipette gun, placing the mixture at 37 ℃ for 30 minutes, immediately placing the mixture on ice for 5 minutes, and carrying out the next step of escherichia coli transformation.

Transformation of Escherichia coli

1. Taking out a tube (100 mu l) of the allelochemicals from a-70 ℃ ultralow temperature freezer, and melting the allelochemicals in ice bath for 5-10 min.

2. Add 5. mu.l of the ligated recombinant plasmid, shake gently and place on ice for 20 min.

3. After shaking gently, the mixture was put into a 42 ℃ water bath for 90 seconds to heat, and then quickly put back into ice and left to stand for 2 min.

4. Adding 500 μ l of non-resistant LB medium into the tubes respectively in a clean bench, mixing, and fixing to a shaker

Shaking the bed on a spring frame at 37 deg.C for 45 min.

5. Taking the conversion mixed solution from a clean bench, pouring the conversion mixed solution into a solid LB plate culture dish containing kanamycin antibiotics,

coating with glass coating rod burned by alcohol lamp, and drying the surface of culture medium in ultra-clean bench.

6. The medium was inverted and placed in a 37 ℃ incubator overnight.

7. Selecting a monoclonal colony, carrying out colony PCR identification, sequencing and then identifying to determine a positive plasmid pOsHEN1 as shown in the specification, namely an OsSPL14 vector.

The pOsHEN1 of the invention comprises an OsSPL14 gene expression box or a recombinant expression vector containing the pOsHEN1 gene expression box, wherein the action mechanism of improving the rice excellent yield traits such as stem thickness and single ear yield so as to improve the rice yield and/or improving the resistance of the rice to rice blight PXO99A is as follows:

rice bacterial blight (Xoo) secretes different effector factors directly into rice cells through a Type three secretion system (T3 SS), and these effector factors bind to interacting targets in rice to generate disease-resistant or disease-susceptible responses. Transcription activation-like (TAL) effector plays a key role as the most important three-type effector of xanthomonas oryzae in the process of interacting with rice. The rice gene OsHEN1 is directly regulated and controlled by Tal9A transcription activation effector of Xoo PXO99A, and the Tal9A transcription activation effector of Xoo PXO99A can be specifically combined with a Tal9A effect combination site of Xoo PXO99A at-25 bp of a promoter of OsHEN1 to activate the promoter of OsHEN 1.

Rice OsSPL14(IPA1) is an ideal plant type gene of rice, the over-expression OsSPL14 reduces the tillering number of the rice, and the expression of the fine control (up-regulation expression) OsSPL14 can improve the yield of the rice by reducing the total tillering number of the rice and increasing the effective tillering number of the rice.

In addition, the inventor unexpectedly discovers that the OsSPL14 plays an important role in the disease resistance of rice for the first time. In rice, the expression of the OsSPL14 gene is up-regulated, and the resistance to bacterial blight can be obviously enhanced.

The constructed pOsHEN1 shows that the OsSPL14 gene expression box is connected with the OsSPL14 gene at the downstream of an inducible promoter OsHEN1, and a rice transgenic line containing the pOsHEN1 gene expression box OsSPL14 gene expression box is obtained after rice is transformed.

The OsHEN1 promoter mainly comprises a target site of a PXO99A pathogenic factor, and meanwhile, most rice material OsSPL14 gene sequences are the same, and sequence differences among individual materials are identical but the functions are the same, so that the technical scheme of the invention can be suitable for various rice varieties.

When the transgenic strain line is not invaded by PXO99A, the expression quantity of background OsSPL14 in the transgenic rice is slightly increased due to the existence of OsSPL14 in the expression cassette, and under the state, the characteristic that OsSPL14 regulates rice tillering can be exerted, tillering can be properly reduced to improve effective tillering number, production consumption is reduced, stem thickness is increased, lodging resistance is improved, rice single-ear yield is improved, and rice yield can be increased through reasonable close planting.

When a transgenic line is invaded by PXO99A, a PXO99A Tal9A transcription activation effector can be specifically combined to a Tal9A effect combination site of Xoo PXO99A at-25 bp on a promoter of OsHEN1, and an OsHEN1 promoter is specifically activated after being induced by Tal9A of Xoo PXO99A, so that the transcription level of a downstream OsSPL14 gene is greatly improved, and the specific enhancement of the disease resistance of PXO99A bacterial blight under the condition of exogenous PXO99A infection is realized.

Meanwhile, the OsHEN1 promoter is used as an inducible promoter, and the inducible promoter has space-time specificity, so that the target OsSPL14 gene can be excessively expressed only at the part infected by PXO99A in a plant body, and the expression quantity in other organs and tissues is not influenced. Therefore, the expression quantity of the OsSPL14 gene in the infected part is greatly increased (increased by dozens of times or hundreds of times), the target resistance is effectively enhanced, the expression quantity of the OsSPL14 gene in other organs and tissues is not affected, and the situation that the tillering number is excessively reduced and the yield is seriously reduced due to the fact that the OsSPL14 of the whole plant is excessively expressed is avoided. The rice yield is not influenced while the disease resistance is effectively achieved, so that the excellent rice variety which can increase the rice yield and enhance the disease resistance to the rice bacterial blight PXO99A is obtained.

The technical scheme of the invention has the following beneficial effects:

after the OsSPL14 gene expression box is transformed into rice, when a transgenic line is not invaded by PXO99A, tillering is properly reduced, so that the effective tillering number is ensured, the production consumption is reduced, the stem thickness lodging resistance is increased, the single-ear yield of the rice is improved, and the yield can be increased through reasonable close planting. When a transgenic line is invaded by PXO99A, the ability of resisting bacterial blight PXO99A can be specifically enhanced at a specific part of rice, the whole OsSPL14 gene expression quantity of the plant is not influenced, and the serious yield reduction caused by excessive reduction of the tillering number is avoided, so that the adverse resistance and development opposition of the rice can be well balanced, a good rice variety which can enhance the disease resistance by increasing the rice yield is obtained, the expression of the OsSPL14 can be accurately regulated and controlled by various breeding means, and the transgenic line has wide application and market prospects in the agricultural field.

Drawings

FIG. 1pCAMBIA1305.1 vector plasmid map and multiple cloning site information schematic.

FIG. 2 shows the strain patterns of four transgenic lines (HS-2, HS-9, HS-8, Hs-4) obtained by infecting transformed rice with the gene expression cassette OsSPL 14.

FIG. 3 four transgenic lines (HS-2, HS-9, HS-8, Hs-4) were upregulated in background OsSPL14 expression without induction by PXO 99A.

FIG. 4 statistics of plant height and tillering of four transgenic lines (HS-2, HS-9, HS-8, Hs-4) under the condition of not being induced by PXO 99A;

in the figure: a is a histogram comparing the plant heights of HS-2, HS-9, HS-8, Hs-4 with wild type NIP; b is a histogram comparing HS-2, HS-9, HS-8, Hs-4 with wild type NIP tillering; n.d. represents a significant level of difference P > 0.05 of the transgenic line compared to the wild-type control; asterisks indicate that transgenic lines differ from wild-type controls by a significant level of P < 0.05; double asterisks indicate that transgenic lines differed from wild-type controls at a significance level of P < 0.01.

FIG. 5 statistics of the third stem thickness of four transgenic lines (HS-2, HS-9, HS-8, Hs-4) without induction by PXO 99A;

in the figure: a is a comparison graph of HS-2, HS-9, HS-8, Hs-4 and a third section of a wild type NIP, b is a comparison histogram of HS-2, HS-9, HS-8, Hs-4 and a third section of the wild type NIP; asterisks indicate that transgenic lines differ from wild-type controls by a significant level of P < 0.05; double asterisks indicate that transgenic lines differed from wild-type controls at a significance level of P < 0.01.

FIG. 6 is a comparison of four transgenic lines (HS-2, HS-9, HS-8, Hs-4) with wild type NIP main spike without induction by PXO 99A;

FIG. 7 is a bar graph comparing four transgenic lines (HS-2, HS-9, HS-8, Hs-4) against wild type NIP branches without induction by PXO 99A;

in the figure: a is a histogram comparing HS-2, HS-9, HS-8, Hs-4 with wild type NIP primary branch; b is a histogram comparing HS-2, HS-9, HS-8, Hs-4 and wild type NIP secondary branches; c is a histogram of the number contrast of HS-2, HS-9, HS-8, Hs-4 and wild type NIP main spike glume flowers; asterisks indicate that transgenic lines differ from wild-type controls by a significant level of P < 0.05; double asterisks indicate that transgenic lines differed from wild-type controls at a significance level of P < 0.01.

FIG. 8 statistics of the number of dominant panicle nodes of four transgenic lines (HS-2, HS-9, HS-8, Hs-4) without induction by PXO 99A;

in the figure: a is a chart for comparing the real numbers of main spikes of HS-2, HS-9, HS-8, Hs-4 and wild type NIP, b is a histogram for comparing the yields of main spikes of HS-2, HS-9, HS-8, Hs-4 and wild type NIP; asterisks indicate that transgenic lines differ from wild-type controls by a significant level of P < 0.05; double asterisks indicate that transgenic lines differed from wild-type controls at a significance level of P < 0.01.

FIG. 9 fluorescent quantitative PCR identification of the expression of OsSPL14 induced by PXO99A in three transgenic lines (HS-2, HS-9, HS-8, HS-4);

FIG. 10 statistics of disease resistance of four transgenic lines (HS-2, HS-9, HS-8, Hs-4) in induced state;

in the figure: a. the length of the lesion is a comparison graph of HS-2, HS-9, HS-8, Hs-4 and wild type NIP lesion length, and b, the length of the lesion is a comparison histogram of HS-2, HS-9, HS-8, Hs-4 and wild type NIP lesion length; c. is a histogram comparing the increasing ratios of HS-2, HS-9, HS-8, Hs-4 resistance; asterisks indicate that transgenic lines differ from wild-type controls by a significant level of P < 0.05; double asterisks indicate that transgenic lines differed from wild-type controls at a significance level of P < 0.01.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The starting materials used in the following examples are commercially available unless otherwise specified, and the detection methods used therein are conventional unless otherwise specified.

Example 1 construction of pOsHEN1:: OsSPL14 Gene expression cassette and construction of recombinant expression vector containing pOsHEN1:: OsSPL14 Gene expression cassette

S1: a clone pOsHEN1-F primer is designed about 2000bp upstream of a transcription initiation site of a rice OsHEN1 gene, a pOsHEN1-R primer is designed at the tail end of a 5' untranslated region of OsHEN1, rice Nipponbare DNA is taken as a template, and an OsHEN1 promoter specific primer pOsHEN 1-F: tatgaccatgattacgaattcTTATGTGCACTAGAAACTATCTGAGGAC (SEQ ID No.1) and pOsHen 1-R: CAAACGCCCAAAAAAAACAA (SEQ ID No.2) to carry out PCR amplification, and cloning out a promoter amplification product of OsHen 1;

s2: a clone OsSPL14-F primer is designed by using a transcription initiation site of OsSPL14, a clone OsSPL14-R primer is designed by about 500bp at the downstream of an OsSPL14 transcription termination site, rice Nipponbare DNA is used as a template, and an OsSPL14 gene specific primer OsSPL 14-F: ttgttttttttgggcgtttgTTCCGTCTCTTTCCTCTCTCTTCT (SEQ ID No.3) and OsSPL 14-R: tggtctttgtagtcaagcttCAGAGACCAATCCATCGTGTTG (SEQ ID No.4), and cloning a genomic DNA amplification product of OsSPL 14;

s3: the promoter fragment of OsHEN1 obtained from S1 and the genomic DNA fragment of OsSPL14 obtained from S2 are connected to the linear fragment of plasmid vector of pCAMBIA1305.1 which is digested by EcoRI and HindIII by a homologous recombination method, and then the vector is transformed by escherichia coli, identified by monoclonal PCR and sequenced to obtain pOsHEN1 in which the genomic DNA fragment of OsSPL14 is connected to the downstream of the promoter fragment of OsHEN1, and the recombinant vector of the OsSPL14 expression cassette.

The homologous recombination system is as follows:

and (3) configuring a recombinant system according to the system, sucking and beating the recombinant system by a pipette gun, uniformly mixing the recombinant system and the pipette gun, placing the mixture at 37 ℃ for 30 minutes, immediately placing the mixture on ice for 5 minutes, and carrying out the next step of escherichia coli transformation.

Transformation of Escherichia coli

1. Taking out a tube (100 mu l) of the allelochemicals from a-70 ℃ ultralow temperature freezer, and melting the allelochemicals in ice bath for 5-10 min.

2. Add 5. mu.l of the ligated recombinant plasmid, shake gently and place on ice for 20 min.

3. After shaking gently, the mixture was put into a 42 ℃ water bath for 90 seconds to heat, and then quickly put back into ice and left to stand for 2 min.

4. Add 500. mu.l each of the above tubes in a clean bench into the non-resistant LB medium and mix gently, then fix on the spring frame of the shaker and shake for 45min at 37 ℃.

5. The above-mentioned conversion mixture was taken out from a clean bench, poured into a solid LB plate culture dish containing kanamycin antibiotic, coated with a glass coating rod burned with an alcohol burner, and the surface of the culture medium was dried in a clean bench.

6. The medium was inverted and placed in a 37 ℃ incubator overnight.

7. Selecting a monoclonal colony, carrying out colony PCR identification, sequencing and then identifying to determine a positive plasmid pOsHEN1 as shown in the specification, namely an OsSPL14 vector.

Example 2 transformation of Rice with recombinant expression vector containing the pOsHen1 Gene expression cassette OsSPL14 to obtain transgenic line HS

The rice used for transformation was: wild type rice Nipponbare (NIP).

A plurality of stable transgenic strains are obtained by using an agrobacterium-mediated transformation method for experimental research of the invention. The specific transformation, infection and transgenic rice culture method comprises the following steps:

1. peeling mature rice seeds, and selecting healthy and complete seeds.

2. Washing and soaking with 70% ethanol for 2-3 min, washing two to three times, and washing with 10% sodium hypochlorite solution for 30 min.

3. Three to four times of sterile water washing.

4. The peeled seeds were dried on sterile filter paper.

5. And (4) placing the blow-dried seeds on an induction culture medium, and culturing for 15-20 days in the dark until the yellow callus grows out.

6. The callus was peeled off and transferred to a subculture medium for two weeks in the dark.

7. And (4) co-culturing. The agrobacterium EHA105 containing the positive plasmid vector and the callus are cultured in a liquid co-culture medium by gentle shaking for 30 minutes, and the co-cultured callus is transferred to sterile filter paper to be dried.

8. The air-dried callus was transferred to a co-culture medium and cultured for 2-3 days.

9. After washing the callus with sterile water containing the antibiotic timentin, the callus was transferred to a selection medium containing 50mg/L hygromycin and cultured in the dark for 2-3 weeks. Transferring to a two-screen culture medium with the concentration of 50mg/L for dark culture until new callus grows out.

10. Transferring new callus to differentiation culture medium, growing rice seedling and transferring to rooting culture medium.

11. Transplanting the seedling to the field when the rooting culture medium grows to be strong.

Four transgenic lines HS-2, HS-9, HS-8, Hs-4 were obtained (FIG. 2).

Example 3 agronomic trait study of transgenic lines not induced by PXO99A

In the research, four transgenic lines HS-2, HS-9, HS-8 and Hs-4 obtained in example 2 are researched for agronomic characters under the condition that the transgenic lines are not induced by PXO99A by taking wild type rice Nipponbare (NIP) as a contrast.

3.1 plant background OsSPL14 expression level

The research method comprises the following steps: fluorescent quantitative PCR

3.2Trizol method for extracting Total RNA

Four transgenic strains HS-2, HS-9, HS-8, Hs-4 and NIP contrast are not induced by PXO99A, and leaf samples are taken to respectively carry out the following operations:

(1) 0.1g of fresh leaf was weighed, quickly ground in liquid nitrogen using a mortar, thoroughly ground and quickly transferred to a 1.5mL centrifuge tube (pre-cooled in liquid nitrogen). Adding 1mL of Trizol into the centrifuge tube, and standing on ice for 10 min;

(2) adding 100uL chloroform, shaking vigorously for 30S, and standing on ice for 5 min;

(3)4℃，13000rpm，10min；

(4) transferring the supernatant into another new 1.5ml RNase-Free centrifuge tube, adding equal volume of isopropanol, gently inverting, mixing, and standing at room temperature for 10 min;

(5)4℃，13000rpm，15min；

(6) discarding the supernatant, adding 500uL RNase-Free 75% ethanol, and washing twice;

(7) pouring off ethanol, and standing at room temperature for about 10 min;

(8) adding 50uL RNase-Free water, keeping the temperature for 15min in a metal bath at 65 ℃, and then preserving at-80 ℃ for later use;

3.3RNA reverse transcription [ HiScript II Q RT Supermix for qPCR (+ gDNA wiper) kit ]

1. Total RNA 1ng-500ng

2.gDNA wiper 2. mu.l

3. Sterile water was added to a total volume of 8. mu.l

The mixture was placed in a 42 ℃ PCR instrument for 2min, and then taken out and placed on ice. Adding 2 microliter HiScript II Q RT SuperMix reverse transcriptase, taking out after 50 ℃ for 15min and 85 ℃ for 2min in a PCR instrument, and diluting by 200 times for later use. 3.4 fluorescent quantitative PCR

Preparation of reaction System

Setting a reaction program:

1. pre-denaturation: 95 ℃ for 5 minutes.

2. And (3) cyclic reaction: 95 ℃ for 10 seconds, 60 ℃ for 30 seconds, 40 cycles.

3. Dissolution curve: 95 ℃ for 15 seconds, 60 ℃ for 30 seconds, 95 ℃ for 15 seconds.

Other detailed steps and fluorescence quantitative PCR are conventional known formulas or common kits in the market, and no special treatment is needed, so that the detailed description is omitted.

Fluorescent quantitative PCR gene number and primer sequence

The results show that: compared with the wild type, the expression of the plant background OsSPL14 of the transgenic plant is up-regulated by 3-15 times under the condition that the transgenic plant is not induced by PXO99A (figure 3).

3.2 plant height and tillering

The plant height and tillering condition of four transgenic lines HS-2, HS-9, HS-8 and Hs-4 are compared with that of wild rice Nipponbare (NIP).

The results show that: compared with the wild plant, the transgenic plant line has increased plant height, reduced tillering number and improved effective tillering number. (FIG. 4)3.3 third Stem thickness

Four transgenic lines HS-2, HS-9, HS-8, Hs-4 were compared with the third stem diameter of wild type rice Nipponbare (NIP).

The results show that: the transgenic lines had increased thickness of the third shoot and increased lodging resistance relative to the wild type (FIG. 5)

3.4 Main spike

Four transgenic lines HS-2, HS-9, HS-8, Hs-4 are compared with the number of primary branches, secondary branches and main spike glume flowers of wild rice Nipponbare (NIP).

The results show that: compared with the wild type first-level branch, the transgenic line has no obvious change, the second-level branch is obviously increased, and the number of the main spike glume flowers is obviously increased (figures 6 and 7)

3.7 number of dominant panicle knots

The main ear number of four transgenic lines HS-2, HS-9, HS-8 and Hs-4 is compared with that of wild rice Nipponbare (NIP).

The results show that: compared with wild type, the yield of the HS-9 and HS-4 main ears of the transgenic line is obviously improved, and HS-2 and HS-8 are not obviously changed. (FIG. 8)

In conclusion, statistics show that the excellent yield traits affecting rice have excellent performance in the transgenic rice HS strain.

Example 4 fluorescent quantitative PCR identification of OsSPL14 specific PXO 99A-induced upregulated expression in HS transgenic lines

In the research, wild type rice Nipponbare (NIP) is used as a control, and the OsSPL14 expression conditions of four transgenic lines HS-2, HS-9, HS-8 and Hs-4 obtained in example 2 after respectively inoculating Xanthomonas albus PXO99A 3 days are researched to determine whether the OsSPL14 is regulated and expressed by the OsHEN1 promoter to be up-regulated under the specific induction of PXO 99A.

The four transgenic lines HS-2, HS-9, HS-8 and Hs-4 are respectively injected to different leaves in the full tillering stage to inoculate the bacterial blight strain PXO99A (OD value is 0.5), the inoculated leaves are sampled at different time points (0h and 72h), the samples in each stage are mixed with three leaves, and total RNA is respectively extracted for identifying the expression of OsSPL 14.

4.1PXO99A germ culture:

PSA culture medium

Peptone 10g/L

Sucrose 10g/L

1g/L sodium glutamate

Agar powder 12g/L

In a sterile super clean bench, the germ liquid is uniformly coated on a PSA culture medium plate, after dark culture at 28 ℃ for 2-3 days, the germ liquid is diluted by sterile water until the bacterium liquid OD600 is 0.5, and the bacterium liquid is used for inoculation experiments.

4.2Trizol method for extracting Total RNA

In 2 inoculation periods after PXO99A inoculation of 3 transgenic strains and NIP control, the following operations are respectively carried out on leaf samples to obtain total RNA in 2 inoculation periods:

(1) 0.1g of fresh leaf was weighed, quickly ground in liquid nitrogen using a mortar, thoroughly ground and quickly transferred to a 1.5mL centrifuge tube (pre-cooled in liquid nitrogen). Adding 1mL of Trizol into the centrifuge tube, and standing on ice for 10 min;

(2) adding 100uL chloroform, shaking vigorously for 30S, and standing on ice for 5 min;

(3)4℃，13000rpm，10min；

(4) transferring the supernatant into another new 1.5ml RNase-Free centrifuge tube, adding equal volume of isopropanol, gently inverting, mixing, and standing at room temperature for 10 min;

(5)4℃，13000rpm，15min；

(6) discarding the supernatant, adding 500uL RNase-Free 75% ethanol, and washing twice;

(7) pouring off ethanol, and standing at room temperature for about 10 min;

(8) adding 50uL RNase-Free water, keeping the temperature for 15min in a metal bath at 65 ℃, and then preserving at-80 ℃ for later use;

4.3RNA reverse transcription [ HiScript II Q RT Supermix for qPCR (+ gDNA wiper) kit ]

1. Total RNA 1ng-500ng

2.gDNA wiper 2. mu.l

3. Sterile water was added to a total volume of 8. mu.l

The mixture was placed in a 42 ℃ PCR instrument for 2min, and then taken out and placed on ice. Adding 2 microliter HiScript II Q RT SuperMix reverse transcriptase, taking out after 50 ℃ for 15min and 85 ℃ for 2min in a PCR instrument, and diluting by 200 times for later use.

4.4 fluorescent quantitative PCR

Preparation of reaction System

Setting a reaction program:

1. pre-denaturation: 95 ℃ for 5 minutes.

2. And (3) cyclic reaction: 95 ℃ for 10 seconds, 60 ℃ for 30 seconds, 40 cycles.

3. Dissolution curve: 95 ℃ for 15 seconds, 60 ℃ for 30 seconds, 95 ℃ for 15 seconds.

Other detailed steps and fluorescence quantitative PCR are conventional known formulas or common kits in the market, and no special treatment is needed, so that the detailed description is omitted.

Fluorescent quantitative PCR gene number and primer sequence

The results show that: within 3 days of inoculation treatment, PXO99A can induce the up-regulated expression of OsSPL14 in an HS transgenic strain, and the result shows that the OsHen1 promoter can regulate and control the downstream OsSPL14 to be significantly up-regulated by 2.75-3.26 times under the specific induction of PXO99A (FIG. 9).

Example 5 study of disease resistance of transgenic lines under induced conditions

5.1 lesion Length

In the research, wild type rice Nipponbare (NIP) is used as a reference, and the lengths of disease spots of four transgenic lines HS-2, HS-9, HS-8 and Hs-4 obtained in the research example 2 are compared after 14 days of respectively inoculating rhizoctonia solani PXO99A and J18 in the tillering stage of rice.

The results show that: in the vigorous tillering stage of rice, for HS transgenic lines and wild type Nipponbare (NIP) leaf-cutting method inoculation strains PXO99A and J18, the result of counting the lesion length shows that the lesion length of PXO99A of the four transgenic lines HS-2, HS-9, HS-8 and Hs-4 in the wild type material is about 1.9cm shorter than that of J18, but the lesion length of PXO99A in the HS transgenic lines is about 3.0-3.8cm shorter than that of J18 (FIGS. 10a and b); research confirms that the HS transgenic line has very strong specific resistance to rice bacterial leaf blight PXO 99A.

5.2 proportion of resistance increase

The study statistics shows that the resistance increase proportion of the four transgenic lines HS-2, HS-9, HS-8 and Hs-4 obtained in the example 2 is compared after 14 days of respectively inoculating the bacterial blight strain PXO99A and J18 in the full tillering stage of rice.

The proportion of increase in resistance: (the length of the wild type PXO99A lesion spot-the length of the HS transgenic strain PXO99A lesion spot)/the length of the wild type PXO99A lesion spot is multiplied by 100% or (the length of the wild type J18 lesion spot-the length of the HS transgenic strain J18 lesion spot)/the length of the wild type J18 lesion spot is multiplied by 100%.

The results show that: the resistance of the HS-2, HS-9, HS-8 and Hs-4 materials of the four transgenic lines to PXO99A is respectively enhanced by 35%, 29%, 32% and 20%, while the resistance of the HS-2, HS-9, HS-8 and Hs-4 materials of the four transgenic lines to J18 is respectively enhanced by about 18%, 13%, 20% and 10%, which is obviously lower than the disease-resistant proportion of PXO99A, and the enhanced resistance of the HS transgenic lines to PXO99A is obviously enhanced by about 60-122% compared with J18, so that the transgenic lines can be determined to be capable of being obviously enhanced in disease resistance under the induction of the specific blight of the white leaf fungus PXO99A (figure 10 c).

Sequence listing

<110> Nanjing university of agriculture

<120> pOsHEN 1:OsSPL 14 gene expression box and construction method and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tatgaccatg attacgaatt cttatgtgca ctagaaacta tctgaggac 49

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

caaacgccca aaaaaaacaa 20

<210> 3

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ttgttttttt tgggcgtttg ttccgtctct ttcctctctc ttct 44

<210> 4

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tggtctttgt agtcaagctt cagagaccaa tccatcgtgt tg 42

<210> 5

<211> 1894

<212> DNA

<213> Rice (Oryza sativa)

<400> 5

gccactgaag taaacaagat gaaaaggaag catgcaggag gggcaggacc cttctggtgg 60

aaatggggga gtaaaaggct actgtacgaa gtgctatttg agggccgctg tctgaggaaa 120

accaaggata caggcaaaga aaaattgaat gatgaagcag agccctaaat gtgttgctgc 180

tgaagcctga ctgctgatct catcaacatc catagtgtag tgggctgact ggggttccct 240

gaatgtagct tgcatatacc gagccaaatt ttgactggag aattaagatg gtagttcgtg 300

tttttaggtg aaatttgtga acaaccttgt tgacatgtat tggatgttca gttgtattag 360

aaatagagtc aagtttacca tcctgtcgtc tgtcgaatga tcctgatgtt catttcaccg 420

cttcatttct gttgcctcgt tatcattttt tatgttaagc ctagcatatg gtattagcaa 480

accagcttga ttgatttttt gcatctctac tggaaattag gaactcaatt gctcatggag 540

ggaagaactc aaagatttgg gattctagct aactttacac agagtccaag ctgtttagga 600

tcctctgttg tgaactgaag agtattaaag agctaaacat gctcttgatc tgcaacagat 660

cagggcagtg ttgatggctc ctctcaccca ctgacccacc atctgtagtt tgttttgatc 720

ttaacctatt catcagcgct ataaagtaga actacggagt actccctcca ttccataata 780

taaggcataa tttatgttcc ataatataaa acatgtatgt atataggtaa ttaactgtga 840

tcccttctcc attaaattat tattttttta aatcctctac ttttatgtta tctaatttta 900

ttggatgcat gcattgtatt tattagaatg atcgaaacta caaaataata ataattattt 960

tcttgatctt ttggttaggg tggttatgcc ttatattttg gaatggaggg agtatttcat 1020

tattaacttc tatgtgcaac taggcaatag catctcagtc catggttttg cttaaactca 1080

cgttagttca gtttcgttaa tgtaaaatcc aatcaagcaa gagtatatgc atactgcatt 1140

cgcaaaaacc acttgaaaaa aaaaactgtg aaatatagag tagtgtttaa cattttactc 1200

ccttcggcca tttgacacaa tagttttaga ccatatagtt tgaccgttgc cttattcaaa 1260

agatttatgt aaatatcact tttttaacct attttgttat taaaattagt ttagtatgac 1320

ttttattttt acatatttgc actaaatttt taaataaaat gttctccctc ttttcttagt 1380

aattaacttt taaaaaatgt ttgaccattt atcttattta aaaattttat gcaaatgcat 1440

atattaaaaa acccagctag tttggtcaaa ctttgatgcg ttaaatattt atatacgagt 1500

gaaacgaccg tatttttcag aaaataatat aacaatcgat ttttacggga cataggcccg 1560

agattttctc aagggacact ttaccctcga aagagattct gcatttccgc taacgttaac 1620

aagaaggcgt attattggat gcagcgccat caaaaccttc tctccccctt cacacactcc 1680

ccctcgcttc ccttccctaa accccacttc acccgactcc tcgaatcctc ctcgcctcgc 1740

ttccgatttt tctggggggt ttagcgcctc ccccaattcg gcgaccaccc cctcccgatt 1800

ccgcctcagt tcctgcgtcg atcgattgcg aaaatcccct tcgttaccaa tcgcatttgt 1860

ttgtttgttt ttgtttgttt tttttgggcg tttg 1894

<210> 6

<211> 4770

<212> DNA

<213> Rice (Oryza sativa)

<400> 6

ttccgtctct ttcctctctc ttctctctcc ccctctcctg gaggagagag aggagaagag 60

gagggggggc cgcgccaaga gccacgcgcg ctacagtctc cttcccaccc gcgaccgcga 120

gcaatggaga tggccagtgg aggaggcgcc gccgccgccg ccggcggcgg agtaggcggc 180

agcggcggcg gtggtggtgg aggggacgag caccgccagc tgcacggtct caagttcggc 240

aagaagatct acttcgagga cgccgccgcg gcagcaggcg gcggcggcac tggcagtggc 300

agtggcagcg cgagcgccgc gccgccgtcc tcgtcttcca aggcggcggg tggtggacgc 360

ggcggagggg gcaagaacaa ggggaagggc gtggccgcgg cggcgccacc gccgccgccg 420

ccgccgccgc ggtgccaggt ggaggggtgc ggcgcggatc tgagcgggat caagaactac 480

tactgccgcc acaaggtgtg cttcatgcat tccaaggctc cccgcgtcgt cgtcgccggc 540

ctcgagcagc gcttctgcca gcagtgcagc aggtcactct ctcactcacc tcgccattgc 600

tgatgtcacc actgcttttg ctttgctttg cttgctctcc ctcctctttc acctatctct 660

cttgtttatt tgcttcttgt tcttgtttag tgctagtaca tgtgttgtta ttgttgtgcc 720

gttttgtctt ttgggttatt gtgttgttgt tactactcgt tttactatag gtttttaagg 780

tttatgagca cggccaccac attagatgca ctgtcaagtg gtgtgtgtgg gacctttcct 840

gctaaaacaa gctgatttca actctctgaa acttcctgca tttcatctat ttttatcttt 900

gattgtgttg ggagtactac actagtagtg ttaatatttt gactggtgct tatgagattt 960

ttaagttggt aggttgatga ggaaaatact cctttatatg gttgagtgat gtgacttgcc 1020

tgtctgcctg cctgcctgcc gctttgcata agattcctct gtgttagtaa gagccactgt 1080

ttatttgtac tggtgcttac tctacttagt taattagcca ttagctataa aattccgttg 1140

atgttgcaag cttagcaatg gccacggtaa gaatgggaga gagaagttgg ctaaagctgt 1200

tgctttgtag tttgtactat atatgtgtct ttgtgttgca agatatgcaa ctcctactat 1260

gctgtgactt gagctcaagg ttttcagtta tctatagatc cttactacta ctgagcatac 1320

taccacttct gtatggtagc atatggtagc atagtccaag ttccaacgcc tcgccagttg 1380

ttcataatct atactaccac ttctgtgcat ttgttacttt tatttaatag tttgtctcat 1440

tagctgacaa gcatatgcct gttttgatat ctgcccctct tgtaatagtc tatggatagc 1500

ttggactgtt tgatgcttta attttttact agcaacactt agggcccctt tgaaatggag 1560

gattagcaaa ggaattttgg aggattcatt ttcctaagga ttttttccta tagagccctt 1620

tgattcatag aaagaggata ggaaaacttc cgtaggattg cattcctatg atcaattcca 1680

taggaaaata agcaagaggt tagacctctt gtgaaacttt cctttgttga gtgtatcttg 1740

tggtataatc aaagggctct tctctccatt tcatgtgttt tcaattcctg taggattgga 1800

aaaacataca acttcaattc ctacgttttt cctattccta tgtttttcct atcctgcgtt 1860

tcaaaggggc ccttaaggat gaagggaagt aagagaaaca tactagagaa tatgtagtag 1920

tatttctaca ttccatattt gtagcactag cccacaaata tctttgcctt gtacttactt 1980

cataccagtt cccccctttt cagagcaaac caacaatttc tgttgcctta tatatctagt 2040

gtcttcgtac taatatatct gttccaaaat gtacctgtcc aaattcatag ctagaaatag 2100

ctttatttag gacggaagta ataactgttg ttagagactt ggttcagact tttggttatg 2160

ttgaggctac tatcatttcc tttacgggcc aaattactac aaatgagaat tcataaaaat 2220

gtcaagattt tatgattgtt gtagctttat ttaggacgga ggtagtaatt gttgttagag 2280

acttggttca gacttttggt tacgttgaag ctactatcat ttcctttatg gtcaaattac 2340

taacaatgag tattcataaa aatgtcaaga ttttataatt gagctgtgcc agtgctaagt 2400

gtgtcactat ctgatgccat aatgcatcat tataaaagcc agatggacca ttagctttta 2460

tgtgtaggac acctgccgtc caattagatg gataaccatc tagtgtttgt gtactgttat 2520

tttaagcccg acatctcaca actccatgaa tgattacagt cttcctttca catggtgtcc 2580

ttttgttgtg ttaggaatag cattttttat ttatgggtgt aattatgaaa ggcactagga 2640

gagttgctgc tttatcttga tgggatttgt agtaatacca tctttaggat gacaagaaat 2700

cttgttctga gttagcatgg gctgcctttt gacctgagct acggtttgct atgtttggct 2760

tgcatcatgc agatctatta ggataataag catataaaag ttgcttgcat tgtgcattgc 2820

ttgttttacc ttgattcatg taggagtaat ttgctcgcca tgcctcgttt tgctttctga 2880

gtcaacagcc aaatttagat gatgtacctt ctgttgcttc aaaaactcag tcactgcaca 2940

gcagcagtgg ataggattca gaatcaatct atccatgatt ctctgttcac ataatatgac 3000

aggttccacc tgctgcctga atttgaccaa ggaaaacgca gctgccgcag acgccttgca 3060

ggtcataatg agcgccggag gaggccgcaa acccctttgg catcacgcta cggtcgacta 3120

gctgcatctg ttggtggtat catcagaggc tcttgttttc tttgcatctt gtgtgtttgt 3180

tggtaactac tggttgcatt cgctgatgtg ttgtttgttg cgattcttga tccagaagag 3240

catcgcaggt tcagaagctt tacgttggat ttctcctacc caagggttcc aagcagcgta 3300

aggaatgcat ggccagcaat tcaaccaggc gatcggatct ccggtggtat ccagtggcac 3360

aggaacgtag ctcctcatgg tcactctagt gcagtggcgg gatatggtgc caacacatac 3420

agcggccaag gtagctcttc ttcagggcca ccggtgttcg ctggcccaaa tctccctcca 3480

ggtggatgtc tcgcaggggt cggtgccgcc accgactcga gctgtgctct ctctcttctg 3540

tcaacccagc catgggatac tactacccac agtgccgctg ccagccacaa ccaggctgca 3600

gccatgtcca ctaccaccag ctttgatggc aatcctgtgg caccctccgc catggcgggt 3660

agctacatgg caccaagccc ctggacaggt tctcggggcc atgagggtgg tggtcggagc 3720

gtggcgcacc agctaccaca tgaagtctca cttgatgagg tgcaccctgg tcctagccat 3780

catgcccact tctccggtga gcttgagctt gctctgcagg ggaacggtcc agccccagca 3840

ccacgcatcg atcctgggtc cggcagcacc ttcgaccaaa ccagcaacac gatggattgg 3900

tctctgtaga ggctgttcca gctgccatcg atctgtcgtc ccgcaaggcg agtcatggaa 3960

ctgaagaacc tcatgctgcc tgcccttatt ttgtgttcaa attttccttt ccagtatgga 4020

aaggaaattc taaggtgact ggcgattaat ctccctgtga tgaataataa tgcgcgccct 4080

tgaactcaat taattgctgt gccgcatcca tctatgtaac tctccatgaa tttttaagta 4140

tcagtgttaa tgctgtattg tcgaggactt ctgctcgata tgttatttct cttatgttgt 4200

tcatcatgaa tctttttctg cttattattc tggtgccggg ttgtccttac cacagaagat 4260

tcagtttcgg ttggcgagag taaacacctt ccctggttgt gacaaaagct ccaacctttt 4320

cacttctcgg cctgtatttg atcttcccct tctgacgctg ttatactact tttaagcctg 4380

tatgtttcca gccttccagg tgaagggcca tactgaagag aaaacatgct ttcagggttt 4440

gatgcattgt gtactttaca agtgtactta agattttgta caatttatat atgtacctgc 4500

tctgctgctg agtattgtag gaaagaatca gttcgaaggg cgtgtgttca tgtaaagtga 4560

gaccacatgc acagcgtgga tttgcagcat gctctctgca ccagtggtgt tctgttgatg 4620

cctttgatgg gctggctgag gtgagaggag gatgatccat gttggcagct tcttcactct 4680

gaaaaataaa agagaagaaa tgttcagatt tgcagacaag tggagagcag tgatatattc 4740

tacaataaaa cattaccacc ttgcttttct 4770

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cccagccatg ggatactact 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcaaagctgg tggtagtgga 20

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aaccagctga ggcccaaga 19

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

acgattgatt taaccagtcc atga 24

Claims (6)

1. The OsHEN1 OsSPL14 gene expression cassette is characterized in that the pOsHEN1 OsSPL14 gene expression cassette comprises an OsHEN1 promoter and an OsSPL14 gene, the OsSPL14 gene is connected with the downstream of the OsHEN1 promoter, the OsHEN1 promoter takes rice Nipponbare DNA as a template, and an OsHEN1 promoter specific primer pOsHEN 1-F: tatgaccatgattacgaattcTTATGTGCACTAGAAACTATCTGAGGAC (SEQ ID No.1) and pOsHEN 1-R: CAAACGCCCAAAAAAAACAA (SEQ ID No.2) was subjected to PCR amplification and cloned.
2. 2. A recombinant expression vector comprising the pOsHEN1 gene expression cassette OsSPL14 as described in claim 1.
3. 3. The recombinant expression vector of claim 2, wherein the recombinant expression vector is obtained by inserting the pOsHen1:: OsSPL14 gene expression cassette of claim 1 between EcoRI and HindIII sites of pCAMBIA1305.1 plasmid vector.
4. 4. The pOsHEN1 of claim 1, wherein the application of the OsSPL14 gene expression cassette or the recombinant expression vector of any one of claims 2 and 3 in improving rice plants comprises: specifically improves the resistance of the new rice variety to the rice bacterial blight PXO99A and/or improves the effective tillering number and/or increases the stem thickness and enhances the lodging resistance.
5. 5. The method of constructing a recombinant expression vector according to claim 2, comprising the steps of:

   s1: taking rice Nipponbare DNA as a template, and taking OsHEN1 promoter specific primers pOsHEN 1-F: tatgaccatgattacgaattcTTATGTGCACTAGAAACTATCTGAGGAC (SEQ ID No.1) and pOsHEN 1-R: CAAACGCCCAAAAAAAACAA (SEQ ID No.2) to carry out PCR amplification, and cloning out a promoter amplification product of OsHEN 1;

   s2: taking rice Nipponbare DNA as a template, and taking an OsSPL14 gene specific primer OsSPL 14-F: ttgttttttttgggcgtttgTTCCGTCTCTTTCCTCTCTCTTCT (SEQ ID No.3) and OsSPL 14-R: tggtctttgtagtcaagcttCAGAGACCAATCCATCGTGTTG (SEQ ID No.4), and cloning a genomic DNA amplification product of OsSPL 14;

   s3: the amplified fragments obtained from S1 and S2 were ligated to EcoRI and HindIII double digested plasmid vector pCAMBIA1305.1 by homologous recombination to obtain a recombinant vector containing the expression cassette of OsHEN1: OsSPL 14.
6. 6. The construction method according to claim 5, wherein the S3 comprises the following specific steps: the promoter fragment of OsHEN1 obtained in S1, the genomic DNA fragment of OsSPL14 obtained in S2 and the linear fragment of pCAMBIA1305.1 after EcoRI and HindIII double enzyme digestion are connected together, transformed by escherichia coli, and subjected to colony PCR identification and sequencing identification after single cloning to obtain the recombinant vector containing the expression cassette of OsHEN1: OsSPL 14.

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