CN117070527A

CN117070527A - Application of rice gene HL6 in regulation and control of diterpene plant protection element content and enhancement of disease resistance

Info

Publication number: CN117070527A
Application number: CN202310977244.4A
Authority: CN
Inventors: 余四斌; 孙文强; 赵志凡; 龙雨林
Original assignee: Hubei Daodao Hongye Biotechnology Co ltd; Huazhong Agricultural University
Current assignee: Hubei Daodao Hongye Biotechnology Co ltd; Huazhong Agricultural University
Priority date: 2023-08-03
Filing date: 2023-08-03
Publication date: 2023-11-17

Abstract

The invention discloses application of a rice gene HL6 in regulating and controlling diterpenoid plant protection element content and enhancing disease resistance, and the expression level of diterpenoid plant protection element synthetic genes (OsCPS 2, osKSL7, osCPS4 and OsKSL 4) in rice is obviously increased after the HL6 gene is knocked out by knocking out the rice HL6 gene (the nucleotide sequence is shown as SEQ ID NO. 1), so that the disease resistance of the rice is improved.

Description

Application of rice gene HL6 in regulation and control of diterpene plant protection element content and enhancement of disease resistance

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a rice gene HL6 in regulating and controlling diterpene plant protection element content and enhancing disease resistance.

Background

Rice is one of the main grain crops in China. Rice production in China is endangered by various diseases such as rice blast, bacterial leaf blight and the like, a large amount of manpower and material resources are needed to be input for carrying out drug control, the rice production cost is increased, the environment is polluted, and the rice eating safety is endangered. Therefore, the cultivation and application of disease-resistant varieties becomes the most economical, safe and effective measure. Diterpene phytochemicals are metabolic products which are synthesized by rice after being infected by pathogenic bacteria and have antibacterial activity, and play an important role in the reaction of resisting various diseases of the rice.

Diterpenoid phytochemicals identified in rice include: rice hull ketones (momisctone) A and B, phytoalexins (phytoalexins) A-F, oryzanol (oryzalexin) A-F and oryzanol S. Diterpene phytochemicals are catalyzed in plastids by 3 molecules of IPP and 1 molecule of DMAPP via GGPP synthase to produce geranylgeranyl diphosphate. GGPP is cyclized into polycyclic olefin under the catalysis of II diterpenoid synthases, then is catalyzed by I diterpenoid synthases to generate a final skeleton, and various modifications are carried out to form diterpenoid plant protection elements of different types. The study shows that the rice genome has 2 diterpene gene clusters which are respectively positioned on the 2 nd chromosome and the 4 th chromosome. The OsCPS4 and OsKSL4 gene clusters located on chromosome 4 are responsible for biosynthesis of rice hull ketone A-B and oryzanol S; the OsCPS2 and OsKSL7 gene clusters located on chromosome 2 are responsible for biosynthesis of phytoalexins A-E and oryzanol A-F.

Researches show that diterpenoid phytochemicals directly participate in disease-resistant reaction of rice, and the diterpenoid phytochemicals mainly participate in disease-resistant reaction by inhibiting the expansion of pathogenic bacteria. After rice plants are infected by pathogenic bacteria such as rice blast, diterpenoid phytochemicals are specifically accumulated at the infected part, and the diterpenoid phytochemicals accumulated in large quantity show strong inhibition activity on spore germination and bud tube growth of the rice blast. OsCPS2 is a diterpene synthase which is compared with upstream, the expression of the OsCPS2 is knocked out or inhibited, the content of diterpene phytoalexin is reduced, the plant is more sensitive to rice blast and bacterial blight, and the content of diterpene phytoalexin is accumulated in a large amount and the disease resistance is enhanced when the OsCPS2 is over-expressed.

HL6 gene encodes a transcription factor containing an AP2 domain that regulates the development of rice leaf and glume coat (Sun et al 2017). However, research reports of HL6 gene regulation of rice diterpenoid phytochemicals for enhancing disease resistance are not yet seen at present.

Disclosure of Invention

The invention aims to provide a novel application of a rice gene HL6, in particular to an application of the knocked-out gene HL6 in improving the content of diterpenoid plant protection elements of rice and enhancing disease resistance.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the application of knocking out the HL6 gene in improving the diterpene plant protection element content and disease resistance of rice is provided, and the nucleotide sequence of the HL6 gene is shown as SEQ ID NO. 1. Specifically, modification of the HL6 coding sequence by CRIPSR-Cas9 gene editing or T-DNA insertion mutation causes the HL6 gene to frame shift, thereby disrupting HL6 protein expression.

A method for creating new rice germplasm with increased diterpene content and disease resistance comprises the following steps: by knocking out the rice HL6 gene (the nucleotide sequence is shown as SEQ ID NO. 1), the HL6 protein expression is destroyed, the expression level of diterpenoid plant protection element synthetic genes (OsCPS 2, osKSL7, osCPS4 and OsKSL 4) is obviously increased after the HL6 gene is knocked out, and the contents of diterpenoid plant protection elements such as rice hull ketone, oryzanol, plant kasheng and the like are obviously increased, so that the disease resistance of the rice is improved.

In the specific embodiment of the invention, rice is transformed by a CRISPSR-Cas 9 vector of a targeted gene HL6, so that 1A base is inserted at the 81bp position of an HL6 gene coding region, a new terminator TAA is generated at the 27-position amino acid position, and the translation of the HL6 protein is stopped in advance, or the frame of the HL6 gene is shifted and stopped in advance by inserting mutation into a T-DNA vector; compared with the wild type, the diterpene plant protection element synthetic genes (OsCPS 2, osKSL7, osCPS4 and OsKSL 4) of the HL6 gene knockout mutant obtained by the two methods have obviously increased expression levels, correspondingly, the contents of diterpene plant protection elements such as plant kasheng D and E, rice hull ketone A and B, rice leaf element C and S and the like are obviously increased, and the resistance enhancement of the HL6 gene knockout mutant to bacterial leaf blight is further verified in a disease resistance experiment.

Drawings

FIG. 1, HL6 Gene editing T0 generation transgenic plants positive identification.

FIG. 2, analysis of mutation sites of HL6 gene editing mutant CR 1.

FIG. 3, identification of the T-DNA insertion mutant of the HL6 gene.

FIG. 4, HL6 gene mutation induces an increase in diterpene synthesis gene expression. A. Comparing the HL6 gene knockout mutant (HL 6-CR 1) with the expression level of the wild ZH11 leaf diterpene plant protection element synthetic gene; B. the expression level of the T-DNA insertion mutant (HL 6-MT) of the HL6 gene is compared with that of a wild type control (HL 6-WT) leaf diterpene plant protection element synthetic gene; * Indicating that the t-test reached a level of 0.01 significantly.

FIG. 5, HL6 gene mutation promotes the increase of diterpenoid plant protection factor content. A. Comparing the HL6 gene knockout mutant (HL 6-CR 1) with the ZH11 leaf diterpenoid plant protection element content; B. comparing the content of diterpene plant protection element in the leaf of the HL6 gene T-DNA insertion mutant (HL 6-MT) with that of diterpene plant protection element in the leaf of a wild type control (HL 6-WT); * Indicating that the t-test reached a level of 0.01 significantly.

FIG. 6 HL6 Gene mutation enhances resistance to white leaf blight. A. Comparing the HL6 gene knockout mutant (HL 6-CR 1) with the length of the bacterial leaf blight post-inoculation spot of the ZH11 leaf; B. the T-DNA insertion mutant of HL6 gene (HL 6-MT) was compared with the length of the bacterial leaf blight post-inoculation lesion of wild type control (HL 6-WT) leaves. The length of the staff gauge is 2 cm; * Indicating that the t-test reached a level of 0.01 significantly.

Detailed Description

Example 1: cloning of Gene HL6

RNA of leaf tissue of flower 11 (Chinese public variety) in rice variety was extracted, reverse transcribed into cDNA, and Polymerase Chain Reaction (PCR) was performed using primers 750F and 750R (primer sequences: ATGGACATGGACATGAGCTCGGCTT and CTACTCCATCCCAAACAAAAAGTTG), PCR procedure: pre-denaturation at 94 ℃ for 5 min; 35 cycles (denaturation at 94 ℃ for 30 seconds; annealing at 55 ℃ for 30 seconds; extension at 72 ℃ for 4 minutes) and extension at 72 ℃ for 10 minutes, sequencing the obtained PCR product to obtain the gene coding sequence of Zhonghua 11HL6, which consists of 1410 bases, and the nucleotide sequence is shown as SEQ ID NO. 1.

The primer is synthesized by Shanghai, and the sequence is determined by Huada genes. RNA extraction and reverse transcription related reagents were purchased from Thermo Fisher, inc., and PCR and reagent formulations were described in molecular cloning Experimental guidelines (J. Sam Brookfield et al, jin Dongyan et al (translation), scientific Press, 2002).

Example 2: construction of HL6 knockout vector and establishment of transformed Agrobacterium

Referring to Tang et al (2018) method, on-line design of HL6 gene knockout vector construction primer HL6-CR-F/R (primer sequences: AGAGGGGCTTCCATCTACAGgttttagagctagaaatagcaagtta and CTGTAGATGGAAG CCCCTCTgccacggatcatctgcacaactc) at Rice Information Gateway (RIGW) website, plasmid pYLsgRNA-OsU3 is used as template, guide sequence (sgRNA) for HL6 gene editing is obtained by PCR, PCR procedure: pre-denaturation at 94 ℃ for 5 min; 35 cycles (denaturation at 94 ℃ for 30 seconds; annealing at 55 ℃ for 30 seconds; extension at 72 ℃ for 1 minute), extension at 72 ℃ for 7 minutes, separating to obtain a sgRNA fragment (AGAGGGGCTTCCATCTACAG) of the HL6 gene, connecting the sgRNA fragment with a pCXUN-Cas9 vector cut by KpnI enzyme, transforming escherichia coli, picking up monoclonal sequencing, selecting a correct mutation-free clone to propagate and extract plasmids, and transforming the constructed HL6 gene knockout vector HL6-CR into agrobacterium EHA105 for transforming flower 11 in rice varieties.

The T-DNA vector pFX-E24.2-15R was transferred into Agrobacterium EHA105 for transformation of flower 11 in rice varieties by the method of Wu et al (2003).

The primers are synthesized by Shanghai, and restriction enzyme KpnI, ligase and agrobacterium EHA105 strain are purchased from Takara company; PCR and reagent formulations are described in the molecular cloning laboratory Manual (J. Sam Broker et al, jin Dongyan et al (translation), science Press, 2002). Sequencing was performed by Huada Gene Inc.

Example 3: agrobacterium-mediated genetic transformation

(1) Induction

Shelling mature flower 11 (Chinese open variety) seed, sequentially treating with 70% ethanol for 1 min, and 0.15% mercuric chloride (HgCl) ₂ ) Seed surface disinfection for 15 minutes; washing the seeds with sterilized water for 4-5 times; placing seeds on a japonica rice induction medium; the inoculated culture medium was placed in the dark for 4 weeks at 25.+ -. 1 ℃.

(2) And (3) subculturing

Selecting bright yellow, compact and relatively dry embryogenic callus, and culturing in dark on non-glutinous rice subculture medium for 2-3 weeks at 25+ -1deg.C.

(3) Pre-culture

Selecting compact and relatively dry embryogenic callus, and culturing in dark on non-glutinous rice preculture medium for 4-5 days at 25+ -1deg.C.

(4) Agrobacterium culture

Pre-culturing agrobacterium on LA medium selected for kanamycin resistance (Shanghai chemical company product) for two days at 28 ℃; the agrobacterium is scraped into a suspension culture medium for suspension culture at 28 ℃.

(5) Infestation of the human body

Transferring the pre-cultured calli into sterilized bottles; regulation of Agrobacterium suspension to OD ₆₀₀ 0.8-1.0; soaking the callus in agrobacterium suspension for 30 min; transferring the callus to sterilized filter paper for drying; then placing on the japonica rice co-culture medium for 3 days at 19-20 ℃.

(6) Screening

Washing the callus with sterilized water for 8 times; immersing in sterilized water containing 400 mg/L Carbenicillin (CN) (Shanghai Ind Co., ltd.) for 30 minutes; transferring the callus to sterilized filter paper for drying; transfer the callus to selection medium of japonica rice containing 250mg/L Carbenicillin (CN) and 50mg/L hygromycin (Hn) (product of Roche Co.) for 2-3 times each for 2 weeks.

(7) Differentiation

The resistant calli were transferred to japonica rice differentiation medium and cultured under light at 26 ℃.

(8) Rooting

Cutting off roots generated during differentiation of the regenerated seedlings; then transferring the strain to rooting culture medium, culturing for 2-3 weeks under illumination, and at 26 ℃.

(9) Transplanting

Residual culture medium on the roots of the regenerated plants is washed off, transferred into pot culture, kept moist for the first few days, and transferred into a field after the plants survive and are strong.

The genetic transformation medium used in this example and the method of preparation thereof are as follows:

(1) Reagent and solution abbreviations

The abbreviations for the phytohormones used for the culture medium in this example are shown below:

6-BA (6-Benzylaminoprone, 6-benzyl adenine)

CN (Carbenicillin )

KT (Kinetin )

NAA (Napthalene acetic acid, naphthalene acetic acid)

IAA (Indole-3-acetic acid, indoleacetic acid)

2,4-D (2, 4-Dichlorophenoxyacetic acid,2, 4-dichlorophenoxyacetic acid)

AS (Acetostingone, acetosyringone)

CH (Casein Enzymatic Hydrolysate, hydrolyzed casein)

HN (Hygromycin B, hygromycin)

DMSO (Dimethyl Sulfoxide )

N6max (N6 macroelement component solution)

N6mix (N6 trace element component solution)

MSmax (MS macroelement component solution)

MSmix (MS microelement composition solution)

(2) Main solution formula

1) N6 medium macroelement mother liquor (prepared according to 10 times concentrated solution (10×)):

the above reagents were dissolved one by one, and then the volume was fixed to 1000 ml with distilled water at room temperature.

2) N6 culture medium microelement mother liquor (prepared according to 100 times concentrated solution (100X))

The above reagents were dissolved at room temperature and fixed to 1000 ml with distilled water.

3) Ferric salt (Fe) ₂ EDTA) stock solution (prepared from 100X concentrated solution)

3.73 g of disodium ethylene diammonium tetraacetate (Na ₂ EDTA·2H ₂ O) and 2.78 g FeSO ₄ ·7H ₂ O is dissolved respectively, mixed and distilled water is used for constant volume to 1000 milliliters, and the mixture is subjected to warm bath for 2 hours at 70 ℃ and is preserved at 4 ℃ for standby.

4) Vitamin stock solution (prepared according to 100X concentrated solution)

Distilled water is added to the mixture to reach 1000 milliliters, and the mixture is preserved at 4 ℃ for standby.

5) MS culture Medium macroelement mother liquor (MSmax mother liquor) (prepared according to 10X concentrate)

The above reagents were dissolved at room temperature and the volume was set to 1000 ml with distilled water.

6) MS culture Medium microelement mother liquor (MSmin mother liquor) (prepared according to 100X concentrated solution)

7) Preparation of 2,4-D stock solution (1 mg/ml):

weighing 100 mg of 2,4-D, dissolving with 1ml of 1N potassium hydroxide for 5 minutes, adding 10 ml of distilled water, completely dissolving, and then fixing the volume to 100 ml, and preserving at room temperature.

8) Preparation of 6-BA stock (1 mg/ml):

weighing 100 mg of 6-BA, dissolving with 1ml of 1N potassium hydroxide for 5 minutes, adding 10 ml of distilled water, completely dissolving, and then fixing the volume to 100 ml, and preserving at room temperature.

9) Preparation of Naphthanoacetic acid (NAA) stock solution (1 mg/ml):

the NAA was weighed 100 mg, dissolved in 1ml of 1N potassium hydroxide for 5 minutes, then dissolved in 10 ml of distilled water to a volume of 100 ml, and stored at 4℃for further use.

10 Preparation of indoleacetic acid (IAA) stock solution (1 mg/ml):

weighing IAA 100 mg, dissolving with 1ml 1N potassium hydroxide for 5 min, adding 10 ml distilled water, and keeping constant volume to 100 ml at 4deg.C.

11 Glucose stock solution (0.5 g/ml):

125 g of glucose is weighed, then distilled water is used for dissolution and volume fixation to 250 ml, and the obtained product is preserved at 4 ℃ for standby after sterilization.

12 Preparation of AS stock solution:

weighing AS 0.392 g, adding 10 ml of DMSO for dissolution, subpackaging into 1.5 ml centrifuge tubes, and preserving at 4 ℃ for later use.

13 1N potassium hydroxide stock solution

Weighing 5.6 g of potassium hydroxide, dissolving the potassium hydroxide in distilled water to a volume of 100 ml, and preserving the potassium hydroxide at room temperature for later use.

(3) Culture medium formula for genetic transformation of rice

1) Induction medium

Distilled water is added to 900 ml, 1N potassium hydroxide is added to adjust the pH value to 5.9, the mixture is boiled and fixed to 1000 ml, the mixture is packaged into 50 ml triangular flasks (25 ml/flask), and after sealing, the mixture is sterilized by a conventional method (for example, sterilization at 121 ℃ for 25 minutes, and the sterilization method of the culture medium is the same as that of the culture medium).

2) Subculture medium

Distilled water is added to 900 ml, the pH value is regulated to 5.9 by 1N potassium hydroxide, the mixture is boiled and fixed to 1000 ml, the mixture is packaged into 50 ml triangular bottles (25 ml/bottle), the bottle is sealed, and the mixture is sterilized according to the method.

3) Pre-culture medium

Distilled water is added to 250 milliliters, the pH value is regulated to 5.6 by 1N potassium hydroxide, the sealing is carried out, and the sterilization is carried out according to the method.

The lysis medium was heated and 5 ml of glucose stock solution and 250. Mu.l of AS stock solution were added before use, and the mixture was poured into petri dishes (25 ml/dish).

4) Co-culture medium

5) Suspension medium

Distilled water is added to 100 milliliters, the pH value is regulated to 5.4, the mixture is packaged into two triangular bottles of 100 milliliters, the triangular bottles are sealed, and the mixture is sterilized according to the method.

1ml of sterile glucose stock solution and 100. Mu.l of AS stock solution were added prior to use.

6) Selection Medium

Distilled water is added to 250 milliliters, the pH value is regulated to 6.0, the sealing is carried out, and the sterilization is carried out according to the method.

The lysis medium was heated prior to use, 250 μl HN (50 mg/ml) and 400 μl CN (250 mg/ml) were added and dispensed into petri dishes (25 ml/dish). And (3) injection: the first selection medium had a carbenicillin concentration of 400 mg/l and the second and subsequent selection medium had a carbenicillin concentration of 250 mg/l.

7) Differentiation medium

Distilled water was added to 900 ml and 1N potassium hydroxide was added to adjust the pH to 6.0.

Boiling, adding distilled water to 1000 ml, packaging into 50 ml triangular flask (50 ml/bottle), sealing, and sterilizing.

8) Rooting culture medium

Distilled water was added to 900 ml and the pH was adjusted to 5.8 with 1N potassium hydroxide.

Boiling, adding distilled water to 1000 ml, packaging into rooting tube (25 ml/tube), sealing, and sterilizing.

Example 4: identification of HL6 Gene knockout mutant

11T 0 generation gene editing plants of HL6 obtained in the example 3 are named CR 1-11 respectively, leaf DNA is extracted, PCR detection is carried out by using Cas9 vector primer CRI-JC-F/R (primer sequences are GAGCGGATAA CAATTTCACACAG and TCTATGTTACTAGATCGGGAATTCA respectively), and the PCR program is 94 ℃ for 5 minutes; 30 cycles (denaturation at 94 ℃ for 30 seconds; annealing at 55 ℃ for 30 seconds; extension at 72 ℃ for 1 minute), extension at 72 ℃ for 7 minutes, and detection of PCR products by 1% agarose gel running, the single plant capable of amplifying a band of about 1000bp was a positive editing plant, and the result is shown in FIG. 1, and 11 plants are all positive transgenic plants. The positive transgenic plants are subjected to PCR amplification sequencing by using primers HL6-CSF/R (primer sequences are ATGAA GTCCATGACGCGGCAGGAGT and TGAAAATCCATGAAAGTCGTACC respectively) at two sides of an editing site of Cas9, and are compared with HL6 sequences of wild medium flowers 11 (ZH 11), wherein 1A base is inserted into the 81 th bp of an HL6 gene coding region of a CR1 plant, a new terminator TAA is generated at 27 amino acids, the translation of HL6 protein is stopped in advance (figure 2), the single seed is harvested for planting T1 generation, CRI-JC-F/R primer detection is continuously utilized, the selfed seed collection of plants which do not contain Cas9 vectors is selected, the designated HL6-CR1, the HL6-CR1 and the wild medium flowers 11 are planted for T2 generation, and the T2 generation seeds of the HL6-CR1 and the ZH11 are mixed.

Example 5: creation of T-DNA insertion mutant of Gene HL6

From the T0 generation T-DNA insertion mutant plants obtained in example 3, leaf DNA was extracted, PCR detection was performed using T-DNA vector primer LBT2 (ATAGGGTTTCGCTCATGTGTTGAGCAT) and HL6 gene primer R1 (TGAAAATCCATGAAAGTCGTACC), wherein the individual strain No. 05Z11DT90 was able to amplify a band, which was a mutant plant in which the T-DNA of the coding region of the HL6 gene was inserted, and selfed seeds of the plant were harvested.

Planting T1 generation of 05Z11DT90 selfed seeds, namely 8 plants, namely M1-M8, extracting leaf DNA, and carrying out PCR detection by using primers F1/R1 (primer sequences are CTAATTTCTCGCCTGTTGGC and TGAAAATCCATGAAAGTCGTACC respectively) on HL6 genes and a T-DNA carrier primer LBT2, wherein the F1+ R1 primer combination expands a band, and a single plant of which the LBT2+ R1 primer combination cannot expand the band is a wild plant without T-DNA insertion; the single plant of which the F1+R1 does not spread the band and the LBT2+R1 spreads the band is a homozygous mutant plant with T-DNA inserted; the individual plants from which the F1+R1 amplified the band and from which the LBT2+R1 also amplified the band were T-DNA inserted heterozygous mutant plants. As shown in FIG. 3, wherein M3 is a wild type single plant, M1, 4 and 5 are positive single plants, the rest are heterozygous single plants, T-DNA homozygous mutant single plant (M4) and wild type single plant (M3) selfing seeds are harvested to plant T2 generation families, M4-derived homozygous T-DNA mutant families are named HL6-MT, M3-derived wild type families are named HL6-WT, and HL6-MT and HL6-WT family seeds are respectively collected in a mixed mode.

Example 6: HL6 mutant and rice diterpenoid phytol synthesis gene expression regulation and control

The method comprises the steps of water planting HL6 gene knockout mutants HL6-CR1, wild flower 11 (ZH 11), HL6T-DNA insertion mutant (HL 6-MT) and isolated wild type control (HL 6-WT), extracting RNA of an aerial part sample of seedlings growing for 10 days, reversely transcribing the RNA into cDNA, and carrying out real-time fluorescence quantitative PCR (primer sequences are shown in table 1) by using specific primers of coding regions of diterpene plant protection element synthetic genes OsCPS2, osKSL7, osCPS4, osKSL4 and internal reference gene Ubiquitin (UBQ), and detecting the expression quantity of the diterpene plant protection element synthetic genes. As shown in FIG. 4, compared with the wild type, the expression level of diterpene plant protection element synthetic genes such as OsCPS2 in the HL6 gene mutant is remarkably increased.

TABLE 1 primer sequences for fluorescent quantitative PCR

Example 7: HL6 mutant and rice diterpenoid phytol content regulation and control

Referring to the method of Zhan et al (2020), the HL6 knockout mutant HL6-CR1, the wild type middle flower 11 (ZH 11) and the HL6T-DNA insertion mutant (HL 6-MT) and the isolated wild type control (HL 6-WT) were hydroponic planted, samples of the aerial parts of seedlings grown for 10 days were taken, ground with liquid nitrogen to powder, 0.1g of the sample powder was placed in a 2mL centrifuge tube, and 4 replicates were set for each material; 1mL of 70% methanol extract was added, vortexed and oscillated for 15s, extracted by ultrasonic waves for 30min, centrifuged at 12000rpm for 10min at 4℃and the supernatant was sucked up, filtered through a 0.22 μm filter membrane to a sample bottle, and diterpene phytochemicals content was measured by using a liquid phase mass spectrometer (model: LCMS-8060). As shown in FIG. 5, the content of diterpene phytochemicals such as plant kasheng D and E, rice hull ketone A and B, oryzanol C and S in the HL6 mutant is remarkably increased compared with the wild type.

Example 8: HL6 mutant and rice bacterial leaf blight resistance regulation and control

Referring to the method of Lu et al (2018), HL6 knockout mutant HL6-CR1, wild type middle flower 11 (ZH 11) and HL6T-DNA insertion mutant (HL 6-MT) and isolated wild type control (HL 6-WT) were grown in a test field. In the heading period of rice, bacterial strain FuJ (bacterial strain of white leaf blight of China) is inoculated by a leaf shearing method, a section with the length of 2-3cm of the tip of each sword leaf is sheared after bacterial solution is dipped by clean scissors, 1-3 sword leaves are sheared by bacterial solution once, 5 sword leaves are sheared by each plant, and 10 plants are sheared by each material. The inoculation situation was investigated after 12d inoculation, the leaf length of the bacterial leaf blight of the disease died was measured, and the statistics of the results are shown in fig. 6, wherein the bacterial leaf blight leaf length of the HL6 mutant is significantly shorter than that of the wild-type control, which indicates that the bacterial leaf blight resistance is enhanced after HL6 mutation.

Reference is made to:

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Claims

1. the application of knocking out the HL6 gene in improving the diterpene plant protection element content and disease resistance of rice is characterized in that the nucleotide sequence of the HL6 gene is shown as SEQ ID NO. 1.

2. The use according to claim 1, wherein said diterpenoid phytochemicals comprise phytocassiae D and E, rice hull ketones a and B, and oryzanol C and S.

3. A method for creating novel rice germplasm with improved diterpene plant protection base content and disease resistance is characterized by knocking out a rice HL6 gene, wherein the nucleotide sequence of the HL6 gene is shown as SEQ ID NO. 1.

4. A method according to claim 3, wherein the rice HL6 gene is knocked out by CRIPSR-Cas9 gene editing.

5. The method according to claim 4, wherein the sgRNA sequence of the rice HL6 gene is shown in SEQ ID NO. 2.

6. A method according to claim 3, wherein HL6 protein expression is disrupted by altering the HL6 coding sequence by T-DNA insertion mutation.