CN117230108B - Application of cabbage BrYUC6 gene in improving heat resistance of plant pollen - Google Patents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
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Abstract
The invention discloses a Chinese cabbageBrYUC6Application of gene in improving heat resistance of plant pollen, wherein the application is over-expression cabbageBrYUC6The gene can improve the resistance of plant pollen at high temperature in early stage of pollen development or inhibit the expression of Chinese cabbageBrYUC6The gene improves the resistance of plant pollen under high temperature stress in late pollen development. CabbageBrYUC6The gene expression change can accurately improve the heat resistance of pollen at different stages of development, and has good application prospect in accurate resistance breeding by utilizing genetic engineering.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of a cabbage BrYUC6 gene in improving heat resistance of plant pollen.
Background
In recent years, with the growth of population and the development of economy, greenhouse effect is more and more prominent, and loss caused by high temperature poses a serious threat to global food safety (Peng et al, 2004;Charles et al, 2010;Chaturvedi etal, 2021). Plants are sessile growing, and are continually challenged by environmental stresses including heat damage, which pose a serious threat to the growth and development of plants (Ding et al 2020). In seed plants, reproductive development is an important process for forming seeds and completing generation alternation, and studies have shown that it is the most sensitive development process to environmental stresses such as high temperature in the plant life cycle, and wherein the sensitivity of male reproductive development (mainly anther and pollen development) to environmental stresses is higher than that of female reproductive development (Begcy et al, 2019). The environmental stress has a serious influence on pollen development, and thus normal production of crop seeds, grains and vegetables taking the seeds as products is affected (De Storme and Geelen,2014;Ghadirnezhad and Fallah,2014). Therefore, the establishment of technical measures against environmental stress or the cultivation of stress-resistant crops is important to ensure the global grain safety.
Pollen development is an important link for the seed plants to finish sexual reproduction and alternate generation, is closely related to agronomic traits such as crop seed yield, quality and the like in agricultural production, has multiple steps and long duration, and can cause different degrees of influence on each period of pollen development due to high-temperature stress (Kim et al, 2001;Giorno et al, 2013; min et al, 2014;Lohani et al, 2020;Chaturvedi et al and 2021). Overall, high temperature stress causes abnormal tapetum degradation times, abnormal pollen grain malformation (shrunken or failed), abnormal pollen wall, no anther dehiscence, premature pollen maturation, pollen germination and abnormal pollen tube growth (Suzuki et al 2001;Lohani et al, 2020;Chaturvedi et al, 2021). Of these, the meiosis to single core microspore stage is considered to be the most susceptible stage to high temperature stress (Sage et al, 2015; masoomi-Aladizgeh et al, 2021; zhang et al, 2022).
The use of plant hormones to combat temperature stress is an important point in the development and basic research of production technology. As an important plant endogenous hormone, auxin has important regulatory effects in anther dehiscence, pollen maturation, filament elongation and pollen tube growth (Cecchetti et al, 2008), and at the same time, its content is extremely susceptible to external environments. Studies in barley and arabidopsis have found that high temperature results in reduced endogenous auxin content of anthers, reduced expression of the auxin synthesis gene YUC6, and exogenous spraying of auxin can improve heat resistance during pollen development (Sakata et al, 2010). However, in cotton, high temperatures cause upregulation of auxin synthesis in temperature sensitive type material anthers, while experiments with externally applied auxin also found that spraying Shi Shengchang at high temperature conditions caused male sterility in heat resistant cotton lines (Min et al, 2014). The difference of the auxin content distribution in different species or the difference of the auxin content in each period of pollen development and the influence of high temperature stress on the auxin in each period is shown, but the related evidence is insufficient.
The biosynthesis of auxins can be divided into two pathways, tryptophan-independent and tryptophan-dependent (Trp) (Zhao et al, 2001). The tryptophan-dependent pathway is currently well studied, and there are 4 pathways (Mashiguchi et al, 2011) such as the YUCC (YUC) pathway, the indole-3-acetamide (IAM) pathway, the Tryptamine (TAM) pathway, and the indole-3-glyoxime (IAOx) pathway (previously referred to as the CYP79B pathway). Among these, the YUC pathway is very conserved in plants, and has been demonstrated in a number of dicotyledonous and monocotyledonous plants to convert the precursor tryptophan to indole-3-pyruvate (IPA) by tryptophan aminotransferase (TAA 1), which in turn catalyzes the formation of auxin via YUC (Mashiguchi et al 2011). The study found that arabidopsis YUC2 was highly expressed early in flower bud development and YUC6 was highly expressed during pollen development, and that double mutations comprising YUC and YUC6 formed pollen-free short stamen, completely losing stamen fertility (Cheng et al, 2006). The flowering phases of arabidopsis and barley are subjected to high temperature stress, which can lead to male sterility, and the expression of auxin synthesis genes YUC and TAA1, etc. is inhibited, especially YUC2 and YUC6, most notably (Sakata et al, 2010). From the above study, the male sterile phenotype of the auxin synthesis deficient YUC2YUC6 mutant is very similar to pollen dysplasia caused by high temperature, suggesting that inhibition of YUC2 and YUC6 expression may be one of the causes of male sterility caused by high temperature. However, current research in crops has focused on high temperature affecting changes in auxin content and related gene expression levels, and there is also a lack of direct evidence for improving plant pollen heat tolerance using mutants or transgenic plants of auxin synthesis pathway genes. Therefore, the potential connection between the cabbage auxin synthetic gene BrYUC6 (the cabbage does not have the homologous gene corresponding to the Arabidopsis YUC 2) and the plant pollen heat resistance is researched by utilizing the modern biotechnology means, which is beneficial to providing theoretical support for taking effective protective measures in propagating the cabbage and other flowering crops to improve the seed yield and quality.
Reference to the literature
Begcy K,Nosenko T,Zhou LZ,Fragner L,Weckwerth W,Dresselhaus T(2019)Male Sterility in Maize after Transient Heat Stress during the Tetrad Stage of Pollen Development.Plant Physiol 181:683-700
Cecchetti V,Altamura MM,Falasca G,Costantino P,Cardarelli M(2008)Auxin regulates Arabidopsis anther dehiscence,pollen maturation,and filament elongation.Plant Cell 20:1760-1774
Charles JH,Godfray JR,Beddington IR,Crute LH,Lawrence.D,F.J,Muir JP,Robinson.S,Sandy M.Thomas,Toulmin C(2010)Food Security:The Challenge ofFeeding 9Billion People.Science 327(5967):812–818
Chaturvedi P,Wiese AJ,Ghatak A,Zaveska Drabkova L,Weckwerth W,Honys D(2021)Heat stress response mechanisms in pollen development.New phytol 231:571-585
Cheng Y,Dai X,Zhao Y(2006)Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis.Genes Dev 20:1790-1799
De Storme N,Geelen D(2014)The impact of environmental stress on male reproductive development in plants:biological processes and molecular mechanisms.Plant Cell Environ 37:1-18
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Disclosure of Invention
The invention provides the application of the cabbage BrYUC6 gene in improving the heat resistance of plant pollen, the expression change of the cabbage BrYUC6 gene can accurately improve the heat resistance of different stages of pollen development, and the application prospect in accurate resistance breeding by utilizing genetic engineering is good.
The technical scheme adopted by the invention is as follows:
the application of the cabbage BrYUC6 gene in improving the heat resistance of plant pollen is that the expression change of the cabbage BrYUC6 gene improves the heat resistance of plant pollen at different stages of development, and specifically comprises the following steps: over-expressing the BrYUC6 gene of the Chinese cabbage improves the resistance of plant pollen at high temperature stress in early pollen development stage, or inhibiting the expression of the BrYUC6 gene of the Chinese cabbage improves the resistance of plant pollen at high temperature stress in late pollen development stage; the over-expressed Chinese cabbage BrYUC6 gene is one or more of the over-expressed Chinese cabbage BrYUC6a, brYUC6b and BrYUC6c genes, the inhibiting expression of the Chinese cabbage BrYUC6 gene is one or more of the inhibiting expression of the Chinese cabbage BrYUC6a, brYUC6b and BrYUC6c genes, wherein the number of the BrYUC6a gene is Braa02g 04530.3C, and the gene sequence is shown as SEQ ID NO. 23; the BrYUC6b gene number is BraA09g005830.3C, and the gene sequence is shown in SEQ ID NO. 1; the BrYUC6c gene is numbered as BraA06g032580.3C, and the gene sequence is shown as SEQ ID NO. 2.
Further, the over-expressed cabbage BrYUC6 gene is an over-expressed cabbage BrYUC6c gene.
Further, the inhibiting expression of the cabbage BrYUC6 gene is inhibiting expression of the cabbage BrYUC6b and BrYUC6c genes.
Further, the plant pollen is dicotyledonous angiosperm pollen or monocotyledonous angiosperm pollen.
Still further, the plant pollen is crucifer pollen.
Further, the plant pollen is cabbage pollen.
Further, when the method is applied, the BrYUC6 gene of the Chinese cabbage or the artificial amiRNA fragment for inhibiting the expression of the BrYUC6 gene is connected into a plant over-expression vector to construct a recombinant over-expression vector, and then the recombinant over-expression vector is transformed into a receptor plant.
Still further, the plant over-expression vector is pCAMBIA1300; the recombinant overexpression vector was transformed into a recipient plant as follows: the recombinant overexpression vector is transformed into agrobacterium, and then the obtained recombinant agrobacterium infects the receptor plant.
The invention has the beneficial effects that:
according to the invention, the BrYUC6 gene of the Chinese cabbage and the artificial amiRNA fragment for inhibiting the expression of the BrYUC6 gene are cloned respectively, genetic transformation is carried out in the Chinese cabbage of the cruciferae, the exogenous BrYUC6 gene of the Chinese cabbage and the artificial amiRNA fragment for inhibiting the expression of the BrYUC6 gene of the Chinese cabbage are expressed in an over-expression manner, so that the heat resistance of pollen at different development stages of pollen can be accurately improved, namely the BrYUC6 gene of the over-expression Chinese cabbage can be improved, the resistance of the BrYUC6 gene of the Chinese cabbage can be inhibited from being improved, and the method has a certain application potential in improving the yield and quality of seeds in the plant seed production process under the high-temperature condition. The gene is applied to resistance breeding in cabbages or other flowering plants, and has good application prospect.
Drawings
FIG. 1 shows the expression analysis of BrYUC6 gene of Chinese cabbage in high temperature treated and untreated control anther of Chinese cabbage. HST represents an anther in tetrad period after high temperature stress (i.e., an anther subjected to high temperature stress in tetrad period), CKT is a control thereof, i.e., an anther in tetrad period grown under normal conditions; HSTM is anther grown to maturity under normal conditions after being subjected to high temperature stress in the pollen development tetrad period, and CKTM is contrast, namely the tetrad grown under normal conditions is developed to maturity anther; HSM is the anther in the late maturation stage of high temperature stress (i.e., the anther in maturation stage subjected to high temperature stress), and CKM is the anther in the maturation stage of its control, i.e., grown under normal conditions.
FIG. 2 shows the BryUC6 gene overexpression (BryUC 6 c) OE ) And inhibition of expression (BrYUC 6 bc) RNAi ) And (5) constructing a vector. A, PCR electrophoresis patterns of CDS clones of BryUC6b and BryUC6c genes of Chinese cabbage. Lanes M are DNA markers, the same applies. B, pCAMBIA1300 vector enzyme digestion electrophoresis diagram. C, cabbage BryUC6 Gene overexpression (BryUC 6b OE And BryUC6c OE ) And (5) positive identification of recombinant vector bacterial liquid. D, cloning PCR electrophoresis pattern of artificial amiRNA fragment (BrYUC 6 bc-amiRNA) for co-inhibiting BrYUC6b and BrYUC6c gene expression of Chinese cabbage. E, cabbage BrYUC6 Gene inhibition expression (BrYUC 6 bc) RNAi ) And (5) positive identification of recombinant vector bacterial liquid. F, cabbage BryUC6 Gene overexpression (BryUC 6c OE ) And inhibition of expression (BrYUC 6 bc) RNAi ) Schematic representation of recombinant vector.
FIG. 3 shows the BryUC6 gene overexpression (BryUC 6 c) OE ) And inhibition of expression (BrYUC 6 bc) RNAi ) And (5) identifying positive plants. A, cabbage BryUC6 Gene overexpression (BryUC 6c OE ) And (5) carrying out PCR identification on the positive plants. The square boxes are three lines selected, the M lane is DNA marker, the W lane is negative control (water), the P is positive control (vector plasmid), and the same is true. B, brYUC6 Gene inhibition expression (BrYUC 6 bc) RNAi ) And (5) carrying out PCR identification on the positive plants. C, hygromycin screening transgenic positive plant schematic diagram. After hygromycin spraying, non-Positive plants yellow or leaves scorch (Negative), and transgenic plants grow well (Positive). D, cabbage BryUC6 Gene overexpression (BryUC 6c OE ) And inhibition of expression (BrYUC 6 bc) RNAi ) Growth conditions of positive plants under normal conditions. CK is a control plant that is not transgenic, i.e., a wild-type control plant.
FIG. 4 shows the BryUC6 gene overexpression (BryUC 6 c) OE ) And inhibition of expression (BrYUC 6 bc) RNAi ) The transgenic plants were observed under normal conditions for pollen germination in vitro (A) and pollen set in vivo (B) (transgenic pollen was applied to the stigma where no transgene (wild type) was performed). OE-12/15/17 is BrYUC6 gene of Chinese cabbageMature pollen under normal conditions of over-expressed (BrYUC 6 c) transgenic lines 12, 15 and 17; RNAi-2/4/8 is the BrYUC6 gene inhibition expression (BrYUC 6 bc) of Chinese cabbage RNAi ) Mature pollen under normal conditions for transgenic lines 2, 4 and 8. CKM is mature pollen under normal conditions for control plants that have not been transgenic. Multiple comparison tests were performed after single factor analysis of variance using different letters (P<0.05 indicates that the sample-to-sample difference is statistically significant.
FIG. 5 shows the BryUC6 gene overexpression (BryUC 6 c) OE ) And inhibition of expression (BrYUC 6 bc) RNAi ) And (5) observing and statistically analyzing the heat resistance of the transgenic strain pollen. A, cabbage BryUC6 Gene overexpression (BryUC 6c OE ) Transgenic plants and non-transgenic control plants were observed under high temperature stress conditions for pollen germination in vitro and pollination in vivo to set seed (transgenic and non-transgenic control treated pollen was fed to the non-transgenic (wild type) stigma), CK was non-transgenic, brYUC6c OE 12/15/17 is a cabbage BrYUC6 gene over-expression (BrYUC 6 c) transgenic line, respectively marked as 12, 15 and 17, HSM is pollen after being subjected to high temperature stress in the mature period, and HSTM is pollen which is recovered to grow to the mature period under normal conditions after being subjected to high temperature stress in the tetrad period. C and D are corresponding statistical analyses (no difference and no statistics), CKM is mature pollen under normal conditions of a control plant which is not subjected to transgenesis, HSTM is pollen which is recovered to grow to mature under normal conditions after being subjected to high temperature stress in a tetrad period of the control plant which is not subjected to transgenesis, and 12/15/17OE-HSTM is pollen which is recovered to grow to mature under normal conditions after being subjected to high temperature stress in a tetrad period of a transgenic line 12, 15 and 17 of Chinese cabbage BrYUC6 gene overexpression (BrYUC 6C). B, brYUC6 Gene inhibition expression (BrYUC 6 bc) RNAi ) Transgenic plants and non-transgenic control plants were observed under high temperature stress conditions for pollen germination in vitro and pollination in vivo to set seed (transgenic and non-transgenic control treated pollen was fed to the non-transgenic (wild type) stigma), CK was non-transgenic, brYUC6bc RNAi -2/4/8 is BrYUC6 gene inhibition expression (BrYUC 6 bc) of Chinese cabbage RNAi ) Transgenic lines, labeled 2, 4 and 8, respectively, HSM being subjected to high temperature stress during maturityAfter the pollen, HSTM is the pollen which is recovered to the mature period under normal conditions after being subjected to high temperature stress in tetrad period. E and F are corresponding statistical analyses (no difference, no statistics), CKM is mature pollen of control plant without transgene under normal condition, HSM is pollen of control plant without transgene after being subjected to high temperature stress in mature period, 2/4/8RNAi-HSM is BryUC6 gene inhibition expression (BryUC 6bc RNAi ) Transgenic lines 2, 4 and 8 pollen after high temperature stress in pollen maturation stage. Multiple comparison tests were performed after single factor analysis of variance using different letters (P<0.05 indicates that the sample-to-sample difference is statistically significant. The scale of the pollen in-vitro germination graph is 100 μm (both the pollen graph A and the pollen graph B share one scale), and the scale of the angle fruit graph is 1cm.
Detailed Description
The invention will be further explained with reference to examples and figures. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention.
EXAMPLE 1 plant Material and high temperature treatment
(1) Cabbage variety 'Byq-97-02' (Brassica campestris L. Ssp. Chinese Makino, syn. B. Rapa ssp. Chinese, brassica campestris syn. B. Rapa) is sown in a matrix of peat, vermiculite and perlite mixed in a mass ratio of 3:2:1, watered in a flowerpot, and dehydrated in a relative humidity of 65% and 300. Mu. Mol.m -2 ·s -1 Culturing in a greenhouse with maximum light intensity until flowering, and photoperiod and temperature of day/night are respectively 16h/8h and 22 ℃/18 ℃.
(2) For high temperature treatment, the cabbage plants grown to the flowering stage under normal conditions (1) are marked (namely, the flowers with the inflorescences of the cabbage are marked in different development periods, 5-grade flower buds are left for testing, the pollen development is typically carried out in 5 periods, namely, pollen blast period, tetrad period, single-core period, double-core period and three-core maturation period (namely, pollen maturation period), and the plants are transferred to a high temperature incubator (Ningbo southeast instruments Co., ltd.) for high temperature treatment at 38 ℃/28 ℃ (day/night 16h/8 h) for 24h, and other conditions are unchanged. The control is to seed the cabbage plant growing under normal condition at the same time. The high temperature treatment was started at 9:00 am. Immediately taking the anther in tetrad period, the anther in mature period and the anther in untreated control tetrad period after high-temperature treatment, and freezing the anther in mature period in liquid nitrogen for gene expression analysis or preserving at-75 ℃ for later use. And (3) recovering the normal condition of the bud in the tetrad period after the high-temperature treatment to grow to the mature period, and taking the anther and the anther in the untreated control mature period, and performing quick freezing in liquid nitrogen for gene expression analysis or preserving at the temperature of minus 75 ℃ for later use. Each treatment treated 10 strains and the test was repeated three times.
EXAMPLE 2 cabbage BryUC6 Gene overexpression (BryUC 6c OE ) And inhibition of expression (BrYUC 6 bc) RNAi ) Vector construction
(1) Extracting total RNA from anther tissue samples of Chinese cabbage at high temperature and in untreated control with different pollen development periods by using Trizol reagent, and completing cDNA synthesis by using TAKARA reverse transcription kit, wherein the specific method comprises the following steps: 5X gDNA Eraser Buffer. Mu.L, gDNA Eraser 1. Mu.L, RNA 1. Mu.g, RNase Free H 2 O is added to 10 mu L,42 ℃ is carried out, the genome DNA is removed for 2min, 5X Primer Script Buffer mu L, RT Primer Mix1 mu L, primer Script RT Enzyme Mix mu L and RNase Free H are added into the reaction solution of the last step 2 O4 mu L, sucking and beating, uniformly mixing, reacting for 20min at 37 ℃, reacting for 5s at 85 ℃ to finish the synthesis of cDNA, and preserving the cDNA at-20 ℃ in a refrigerator.
(2) qRT-PCR analysis was performed on the expression characteristics of the white cabbage BrYUC6 gene (three copies in the cabbage, brYUC6a, brYUC6b and BrYUC6c, numbered Braa02g04530.3C, braa09g005830.3C, braa06g032880.3C, respectively, as shown in SEQ ID NO.23, SEQ ID NO.1, SEQ ID NO. 2) in different pollen development stages using the cabbage anther cDNA of (1) as a template and the primers of Table 1 (designed by Primer Premier 5). The qRT-PCR reaction system was 15. Mu.L: 7.5 mu L of SYBR Green Master Mix, 0.3 mu L each of forward and reverse primers, 1 mu L template, 5.9 mu L ddH 2 O. qRT-PCR reaction scheme: 95 ℃ C:: 30s,40 cycles (95 ℃ C.: 5s,55 ℃ C.: 45 s). The specificity of the reaction was determined by melting curve, the reference gene was BrUBC10, and the relative expression level of the gene was 2 -ΔΔCt And (5) calculating a method.
The results indicate that early pollen development (represented by tetrad) was subjected to high temperature stress such that BrYUC6 gene expression was decreased, but late pollen development (represented by pollen maturation) was subjected to high temperature stress such that its expression was increased (fig. 1).
TABLE 1 primers for qRT-PCR analysis of BrYUC6 genes of Chinese cabbage
| Primer name | Primer sequence (5 '-3') |
| BrYUC6c-qRT-F | TTCTGCGAGCTTCCGCTTAT |
| BrYUC6c-qRT-R | TCGACCGTCTGACCAAACTC |
| BrYUC6b-qRT-F | TGAGGACTACGCCAAGAGGT |
| BrYUC6b-qRT-R | CCAACCACCGGCAAACATAC |
| BrYUC6a-qRT-F | GCTCAGCCTTCTCTCGTTGT |
| BrYUC6a-qRT-R | CAACGAGTTGGATGGGCAAC |
| BrUBC10-F | GGGTCCTACAGACAGTCCTTAC |
| BrUBC10-R | ATGGAACACCTTCGTCCTAAA |
(3) According to the result in (2), selecting BrYUC6B and BrYUC6C genes with higher expression in anther tissue, carrying out PCR amplification by using primers with pCAMBIA1300 vector homology wall in table 2 to obtain PCR products (figure 2A), obtaining BrYUC6B and BrYUC6C gene clones with vector homology wall, carrying out rubber cutting recovery on the target strips, carrying out tangential pCAMBIA1300 vector (figure 2B) by using restriction enzymes BamH I and Sac I, carrying out rubber cutting recovery, carrying out recombination on BrYUC6B and BrYUC6C gene fragments and linearized vector fragments by using a homologous recombination method, carrying out thermal shock conversion on the recombined products, randomly picking single colony, carrying out PCR identification by using primers in table 3 (figure 2C), picking positive extracted colony, and carrying out sequencing verification by sequencing company (Zhejiang Kagaku biological science and technology Co., ltd.) to obtain BrYUC6B OE Recombinant plasmid and BryUC6c OE Recombinant plasmid (FIG. 2F), -storing at 20℃for further use.
The specific steps involved are as follows:
(1) fragment amplification
PCR System 50. Mu.L: 1. Mu.L of DNA/cDNA (template), 25. Mu.L of 2X Phanta Max Master Mix (high fidelity enzyme premix), 2. Mu.L of forward and reverse primers, ddH, respectively 2 O 20μL。
PCR procedure: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15s, annealing at 58℃for 15s (the temperature can be adjusted according to the designed primer), extension at 72℃for 1min (the effect of the enzyme and the fragment size are adjusted), and total extension at 72℃for 5min.
The amplified product was spotted into 1.0% of the mesoporous gel and run for 30min at 120v and 100mA in an electrophoresis apparatus, and photographed in a gel imager.
(2) Enzyme cutting
The system comprises: 1. Mu.g of DNA plasmid, 2. Mu.L of BamH I and Sac I enzymes each, 4. Mu.L of Buffer, ddH 2 O was made up to 20. Mu.L.
The reaction: the reaction was carried out at 37℃for 1h.
The product after enzyme digestion was spotted into 1.0% of the mesoporous gel and run for 30min in an electrophoresis apparatus at 120v and 100mA, and photographed in a gel imager.
(3) Gel recovery
Cutting off a gel block containing a target strip under ultraviolet light, placing the gel block into a 1.5mL centrifuge tube, adding about 300 mu L (the gel block can be completely immersed, and 300 mu L of the gel block is added into each 0.1g of gel), placing the gel block into a 37 ℃ metal bath/water bath, and heating to enable the gel block to melt and release a target fragment (about 5-10 min); transferring the solution into DNA Fragment Mini column (namely a kit matched column), and standing for 1min at room temperature; centrifuging at 12000rpm for 1min, and discarding filtrate; 750ml Buffer WB (the absolute ethyl alcohol with the specified volume is added in advance) is added into the Mini column, the mixture is centrifuged for 1min at 12000rpm, the filtrate is discarded, and the process is repeated once; mini column was mounted on new 2mL Collection Tube and centrifuged at 12000rpm for 2min; mini column was placed on a new 1.5mL centrifuge tube and 50. Mu.L ddH was added to the center of the Mini column membrane 2 O (heating to 50-65 ℃ to improve DNA eluting efficiency), standing for 1min at room temperature; centrifuging at 12000rpm for 2min to obtain purified fragments. The concentration was then measured and stored at-20 ℃.
(4) Homologous recombination
The system comprises: about 60ng of gene fragment (adjusted according to fragment size, generally about 0.04. Times. Total base pair number of fragments), 5 XCE MultiS Buffer 4. Mu.L, exnase MultiS 2. Mu.L, ddH 2 O was replenished to 20. Mu.L.
The reaction: the reaction was carried out at 37℃for 30min.
(5) Conversion by heat shock
Thawing DH5 a E.coli on ice; sucking 10 mu L of homologous recombination product, adding into 100 mu L of escherichia coli, gently sucking and beating, and uniformly mixing; standing on ice for 30min; heat shock is carried out on the metal bath at the temperature of 42 ℃ for 60 seconds, and then the metal bath is immediately placed on ice for cooling for 2-3 minutes; adding 1mL of LB liquid medium without any antibiotics into an ultra-clean bench, and putting into a shaking table at 37 ℃ for resuscitation for 45-60min; centrifuging at 5000rpm for 2min, and discarding most filtrate to leave about 100 μl; mixing the rest filtrate and fungus blocks with a pipetting gun; the bacterial liquid was uniformly spread on LB solid medium containing antibiotic (kanamycin 50 mg/L), the plate was sealed with a sealing film, marked, and placed in an incubator at 37℃for overnight culture.
(6) Colony positive identification
8 single colonies were randomly picked up in the solid medium of each recombinant vector in (5) on a super clean bench, and were shaken on a shaker at 37℃for 4-5h in 1.5mL centrifuge tubes with 1mL LB liquid medium containing kanamycin (kanamycin 50 mg/L).
PCR System 25. Mu.L: 1. Mu.L of bacterial solution (template), 1. Mu.L of 2 XRapid Taq Master mix 12.5. Mu.L of forward and reverse primers, and ddH 2 O 9.5μL。
PCR procedure: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15s, annealing at 58℃for 15s (the temperature can be adjusted according to the designed primer), extension at 72℃for 1min (the effect of the enzyme and the fragment size are adjusted), and total extension at 72℃for 5min.
The amplified product was spotted into 1.0% of a gel in an electrophoresis apparatus at 120v for 30min at 100mA, and photographed in a gel imager.
(7) Plasmid extraction
Adding 100 mu L3M NaOH,12,000rpm into the adsorption column, centrifuging for 30-60s, and carrying out column balancing; (6) centrifuging the medium positive bacterial liquid at 5000rpm for 1min, and discarding the supernatant; adding 250 mu L of Solution I, reversing and uniformly mixing to fully re-suspend the thalli; adding 250 μLSolution II, gently reversing the above and below for several times, and standing for 2-3min; 350. Mu.L Solution III was immediately inverted up and down several times to produce a precipitate; centrifuging at 12000rpm for 10min; transferring the supernatant into an adsorption column after balancing, centrifuging at 12000rpm for 1min, and discarding the waste liquid; adding 500 mu L of HBC buffer, centrifuging at 12000rpm for 1min, and discarding the waste liquid; adding 700 μL of Wash Buffer, centrifuging at 12,000rpm for 1min, discarding the waste liquid, and repeating for one time; centrifuging at 12,000rpm for 2min, and discarding the waste liquid; the tube was replaced with 1.5mL centrifuge tube and received, and 50. Mu.L ddH was added 2 O, standing for 1min at room temperature; centrifuging at 12,000rpm for 1min; after the concentration was measured, the samples were stored at-20 ℃.
TABLE 2 PCR amplification primers for CDS sequences of BryUC6b and BryUC6c Gene with vector homology arms of Chinese cabbage
TABLE 3 identification primers for positive colonies of BryUC6b and BryUC6c gene recombinant vectors of Chinese cabbage
| Primer name | Primer sequence (5 '-3') |
| 35S-1300GFP-F | CTCCTCGGATTCCATTGCCC |
| BrYUC6b-R | TTCTAGTAATCTACTGTAGGGTTGACCA |
| BrYUC6c-R | GGCTTGATCAGGTTTACTTGACATG |
(4) According to the CDS sequences of the BrYUC6b and BrYUC6c genes of the Chinese cabbage obtained in the step (3), designing and screening artificial miRNA fragments (which can not be designed to inhibit three genes simultaneously) for co-inhibiting the BrYUC6b and the BrYUC6c by utilizing an online network tool (http:// wmd3.Weigelworld. Org /) and (https:// www.zhaolab.org/psRNATarget /), selecting BrYUC6b and BrYUC6c with relatively high expression in anther tissues and low difference multiple after high temperature stress, comparing the initially screened fragments in a Chinese cabbage database (http:// brassicadb. Cn), finally determining the preferred artificial miRNA fragments (bc-amiRNA) for specific co-inhibition of the BrYUC6b and the BrYUC6c, and delivering the obtained product to a biological company (Beijing department biology)Sciences Co., ltd.). PCR amplification was performed by high fidelity enzyme using primers with pCAMBIA1300 vector homology wall in Table 4 to obtain PCR products, thus obtaining co-suppressed BrYUC6b and BrYUC6c artificial miRNA (BrYUC 6 bc-amiRNA) clones with vector homology wall. Constructing recombinant plasmid according to the specific method in (3) and subjecting the recombinant plasmid to sequencing verification by a sequencing company to obtain BrYUC6bc RNAi The recombinant plasmid is preserved at-20 ℃ for standby. The results are shown in FIGS. 2D-F.
TABLE 4 primers for PCR amplification of BrYUC6bc-amiRNA with vector homology wall
| Primer name | Primer sequence (5 '-3') |
| BrYUC6bc-amiRNA-p1300-F | gagctcggtacccggggatccGGGTGAGAATCTCCATGTTCGT |
| BrYUC6bc-amiRNA-p1300-R | gcccttgctcaccatgtcgacGGGTGAAGAGCTCATGTTCGTAT |
Table 5BrYUC6bc-amiRNA recombinant vector positive colony identification primers
| Primer name | Primer sequence (5 '-3') |
| BrYUC6bc-amiRNA-F | GGGTGAGAATCTCCATGTTCGT |
| 35S-1300GFP-R | AACTTGTGGCCGTTTACGTC |
Example 3 genetic transformation of cabbage Using vacuum flower dipping
(1) Recombinant over-expression vector for transforming agrobacterium
From the previous results (FIG. 1), it can be seen that the three copies of BrYUC6 (BrYUC 6a, brYUC6b and BrYUC6 c) of cabbage have similar expression tendencies before and after high temperature treatment, and that the over-expressed BrYUC6c of BrYUC6c, which is the highest in terms of pollen development, obtained in example 3 is selected due to the multi-copy functional redundancy phenomenon in cabbage OE And co-inhibited BrYUC6b and BrYUC6bc of BrYUC6c RNAi The recombinant plasmid is transferred into agrobacterium GV3101, and the specific method is as follows: mixing 50. Mu.L of thawed Agrobacterium competent cells with 1. Mu.g of recombinant plasmid obtained in example 2, standing on ice for 10min, reacting in liquid nitrogen for 5min at 28deg.C, and standing on ice for 5min; adding 1mL of liquid LB culture medium without any antibiotics on an ultra-clean workbench, and culturing for 4-5h at a temperature of 28 ℃ by using a shaking table at 200 rpm; centrifuging at 5000rpm,2min, discarding most supernatant, suspending the cell mass about 100 μl, and applying to a medium containing rifampicin (50mg.L) -1 ) Kanamycin (50 mg.L) -1 ) Solid LB plates; and (3) culturing for 2 days in an inverted mode after forward standing at 28 ℃ for 30min. After the PCR detection of the positive colony is correct, the strain is re-suspended by 30v/v% glycerol LB, namely the strain contains BrYUC6c OE Agrobacterium GV3101 strain of recombinant plasmid and BryUC6bc containing strain RNAi The recombinant plasmid Agrobacterium GV3101 strain is preserved at-75 deg.c separately for further use.
(2) Vacuum flower dipping method for converting Chinese cabbage
Two days before infection, the product containing BryUC6c is taken out from the ultra-low temperature refrigerator OE Agrobacterium GV3101 strain of recombinant plasmid and BryUC6bc containing strain RNAi The agrobacterium GV3101 strain of recombinant plasmid is inoculated onto the super clean bench with inoculating loopSeed inoculated in a medium containing rifampicin (50 mg. L) -1 ) Kanamycin (50 mg.L) -1 ) On solid LB screening plates, activated species. After inoculation, the cells were sealed, inverted and incubated in an incubator at 28℃for 36h. Single colonies were picked and grown to 15mL containing rifampicin (50 mg. L) -1 ) Kanamycin (50 mg.L) -1 ) In LB liquid medium of (C). Placing into a shaker at 28deg.C, culturing at 200rpm for 12 hr to obtain agrobacterium mother liquor, and preserving at 4deg.C. The day before infection, 5mL of mother liquor was taken in 500mL of liquid LB medium (containing rifampicin (50 mg. L) -1 ) Kanamycin (50 mg.L) -1 ) 28℃until the OD value is 0.8-1.0.
Removing flowering flowers of the cabbage after bolting and flowering on the day of infection, and stripping buds of unopened flowers (only the stigmas are exposed); the bacterial liquid was centrifuged at room temperature (4000 rpm,10 min), the waste liquid was discarded, 500mL of 5wt% sucrose solution was added to resuspend the bacterial cells, 200. Mu.L/L of the surfactant Silwet-77 was added, and the mixture was stirred uniformly. Completely immersing the bud-removed cabbage inflorescence in agrobacterium resuspension, vacuumizing for 5min under the pressure of-0.09 MPa, deflating for 5min, vacuumizing for 5min under the same conditions, sucking excessive agrobacterium resuspension by using water-absorbing paper, dark culturing for 48h in a moist and dark environment, pollinating the plant immersed with the pollen of the plant immersed with the pollen, and teaching again every 1 day for 2 weeks. Culturing for about 1 month to obtain seeds.
(3) Screening and detecting transgenic positive Chinese cabbage plants
Primary screening of hygromycin: sowing the cabbage seeds treated in the step (2) into a matrix (formed by mixing peat, vermiculite and perlite according to a mass ratio of 3:2:1), and using 200 mg.L in the morning after germination -1 The hygromycin is sprayed (liquid drops out of leaf surfaces) for one week, then the growth condition of the seedlings is observed, and the seedlings with the minimum leaf scorch area or without scorch are selected for further PCR detection.
And (3) PCR detection: the seedlings were first screened and 1cm was taken 2 The leaves were subjected to DNA extraction. DNA extraction: extracting DNA by simple extraction method, firstly preparing DNA extraction buffer solution, taking 20wt% SDS solution 0.5ml,0.5 mol.L -1 EDTA aqueous solution 0.5mL,1 mol.L -1 Tris-HCl buffer(pH 9.0)2mL，2mol·L -1 LiCl solution 2mL, double distilled water is added to 10mL, and the mixture is uniformly mixed for use. Then, about 0.1g of the sample is put into a 2mL centrifuge tube, 200 mu L of DNA extraction buffer solution and 1 magnetic bead are added, the mixture is put into a sample grinder for grinding (65 Hz,120 s), and after the magnetic beads in the centrifuge tube are taken out, the mixture is centrifuged at 12000rpm for 5min; transferring 100 mu L of supernatant to a new 1.5mL centrifuge tube, adding 100 mu L of isopropanol, rapidly and gently reversing and uniformly mixing, standing at room temperature for 5min, and centrifuging at 13,000rpm for 10min; removing the supernatant, washing the precipitate with 1mL of 70 v/v% ethanol, centrifuging at 12000rpm for 3min, and removing the supernatant; after repeated washing, the centrifuge tube was inverted on absorbent paper, and after drying the ethanol, 50. Mu.L of double distilled water was added to dissolve DNA. The resulting DNA was detected by PCR, and specific steps and primers used in (3) and (4) of example 2 were described.
The results are shown in FIG. 3.
Example 4 phenotypic observations and statistical analysis
(1) In vitro germination test
The transgenic plants obtained in example 3 and the control plants not subjected to the transgene (wild type) were treated and grown under normal conditions in accordance with the planting and high temperature treatment method in example 1. Mature pollen of transgenic plants under normal conditions and control plants without transgenesis, transgenic plants after high temperature treatment and mature pollen of control plants without transgenesis (pollen after high temperature stress in maturation period and pollen which is recovered to grow to maturation period under normal conditions after high temperature stress in tetrad period) are taken and cultured in 30 mu L of culture medium (15 wt% sucrose, 0.4 mmol.L) -1 HBO 3 、0.4mmol·L -1 Ca(NO 3 ) 2 And 0.1wt% agar, with 2 mmol.L -1 NaOH solution to adjust pH to 5.8), culturing for 3 hours at 20deg.C in dark condition, respectively dripping 30 μl Alexander dye solution (for detecting pollen viability), staining for 30min at room temperature in dark condition, observing under microscope, photographing, and analyzing the picture with imageJ software.
(2) In vivo hybridization set-up test
Mature pollen of a transgenic plant and a control plant which is not subjected to transgenesis, the transgenic plant and the mature pollen of the control plant which is not subjected to transgenesis (pollen after the mature period is subjected to high temperature stress and pollen which is recovered to grow to the mature period under the normal condition after the tetrad period is subjected to high temperature stress) are taken as male parents, the plant which is not subjected to transgenesis under the normal condition is taken as female parents, pollination and growth are carried out under the normal condition, and the fruiting condition of the cone fruits is observed for about 20 days.
(3) Statistical analysis
These experimental results were statistically analyzed and plotted using Graphpad prism 9.0 (GraphPad Software, san Diego, USA) software. Data are expressed as mean ± SD and further evaluated using analysis of variance to determine significance. At a 95% confidence level, the difference was considered significant (p < 0.05).
As a result, as shown in FIG. 4, although fertility of the transgenic plants was reduced under normal conditions, the cabbage was a whole-flower inflorescence and had a large pollen number, so that it had little effect on seed production. As shown in FIG. 5, the BrYUC6c over-expression transgenic plant of Chinese cabbage can improve pollen resistance under high temperature stress at early pollen development (represented by tetrad period) and inhibit expression of BrYUC6bc RNAi Transgenic plants are capable of increasing pollen resistance under high temperature stress in late pollen development (represented by pollen maturation). The BrYUC6 genes of the Chinese cabbage and the artificial amiRNA fragments for inhibiting the expression of the BrYUC6 genes are respectively overexpressed in the receptor plants, so that the heat resistance of different stages of pollen development can be accurately improved, and precious resources are provided for breeding new varieties of plant resistance.
While the foregoing embodiments have been described in detail in connection with the embodiments of the invention, it should be understood that the foregoing embodiments are merely illustrative of the invention and are not intended to limit the invention, and any modifications, additions, substitutions and the like made within the principles of the invention are intended to be included within the scope of the invention.
Claims (4)
1. The application of the cabbage BrYUC6 gene in improving the heat resistance of plant pollen is characterized in that: the BrYUC6c gene of the Chinese cabbage is overexpressed to improve the resistance of plant pollen at high temperature stress in early pollen development; wherein the BrYUC6c gene is numbered as BraA06 g032580.3C, and the gene sequence is shown in SEQ ID NO:2 is shown in the figure; the plant pollen is cabbage pollen; the early stage of pollen development is a tetrad stage of pollen development.
2. The application of the cabbage BrYUC6 gene in improving the heat resistance of plant pollen is characterized in that: co-inhibiting expression of BrYUC6b and BrYUC6c genes to increase plant pollen resistance under high temperature stress at late pollen development; wherein the BrYUC6b gene is numbered as BraA09g005830.3C, and the gene sequence is shown in SEQ ID NO:1 is shown in the specification; the BrYUC6c gene is numbered as BraA06g032580.3C, and the gene sequence is shown in SEQ ID NO:2 is shown in the figure; the plant pollen is cabbage pollen; the late pollen development stage is pollen maturity stage; wherein, the nucleotide sequence of the artificial microRNA fragment for jointly inhibiting the expression of BrYUC6b and BrYUC6c genes is shown in SEQ ID No: 3.
3. The use according to any one of claims 1-2, wherein the cabbage BrYUC6c gene or an artificial miRNA fragment co-inhibiting expression of BrYUC6b and BrYUC6c genes is linked to a plant over-expression vector, constructed to obtain a recombinant over-expression vector, and the recombinant over-expression vector is then transformed into a recipient plant.
4. The use according to claim 3, wherein the plant over-expression vector is pCAMBIA1300; the recombinant overexpression vector was transformed into a recipient plant as follows: the recombinant overexpression vector is transformed into agrobacterium, and then the obtained recombinant agrobacterium infects the receptor plant.
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