CN109762840B - Application of over-expression Chinese cabbage MYB55 in cabbage type rape molecular breeding - Google Patents

Abstract

The invention discloses application of over-expression Chinese cabbage MYB55 in cabbage type rape molecular breeding, wherein a positive transgenic plant is obtained after a plus 10 number in a black-seeded cabbage type rape variety is transformed by constructing a sense over-expression plant vector; the transgenic positive plants are obviously lagged in development, the height of the plants is obviously lower than that of the control plants before full-bloom, but the transverse growth of the transgenic plants is developed; reproductive growth of the transgenic plants is delayed by 3-5 days compared to the control wild type plants, but flower, bud and silique numbers are increased, so that the yield of the whole transgenic plants is increased. Tissue anatomy observation of the transgenic plant shows that the stem vascular bundle of the transgenic plant is enlarged, the xylem is thickened, the area is larger, and the bending resistance is increased; in addition, sclerotinia is inoculated to the stem of the transgenic plant, the disease spot is obviously smaller than that of a wild plant, and the result shows that the BrMYB55 participates in the growth and development of the plant, increases the yield and participates in the processes of lodging resistance and sclerotinia rot resistance of the plant.

Description

Application of over-expression Chinese cabbage MYB55 in cabbage type rape molecular breeding

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of over-expression Chinese cabbage (Brassica rapa) MYB55 in cabbage type rape molecular breeding, which can improve the growth and development of cabbage type rape, increase yield, resist lodging and disease.

Background

Chinese cabbage originates from China, is one of the most important vegetables and oil used as a substance in China, and has a complex genetic basis, so that improvement of some important agronomic traits is very difficult. A plurality of researches show that the limited material types and the limited mark quantity lead the genetic diversity research of the Chinese cabbage crops to be incomplete, and the morphological physiological characters and the nutritional quality characters such as phytic acid containing children and the like lack the systematic analysis of the genetic rule. It is necessary to discuss the genetic variation of related characters on the molecular level, and lays a foundation for discovering and utilizing key gene sources.

Chinese cabbage belongs to the family Brassicaceae, and the model plant Arabidopsis also belongs to the family Brassicaceae. Therefore, the research result of the model plant Arabidopsis thaliana functional genomics can promote the molecular mechanism research of important characters of the Chinese cabbage. Currently, the MYB55gene is not studied comprehensively and profoundly throughout the plant community. The previous research finds that the expression of the Arabidopsis AtMYB55 in leaf basal cells is relatively high, and the AtMYB55 is presumed to be related to leaf morphogenesis; the study by Allison Gaudinier et al found by yeast single-hybrid technology that AtMYB55 interacted with AtIRX11 in the root column and roots, whereas AtIRXII gene was shown to be up-regulated by SND1 and MYB46, so it was speculated that AtMYB55 might be involved in the biosynthesis of the secondary cell wall; the report by Hideki Goda et al shows that AtMYB55 is regulated by auxin and brassinolide in Arabidopsis thaliana. All the above studies indicate that AtMYB55 can improve growth and development of arabidopsis thaliana. In addition, Jianweii Zhao et al found that treatment of oilseed rape with sclerotinia led to significant upregulation of MYB55, suggesting that MYB55 is involved in the sclerotiniose resistance process in oilseed rape. However, at present, the membership number, protein characteristics, evolutionary relationship, expressed tissue specificity, relationship with growth and development, yield increase, lodging resistance and disease resistance, application in genetic engineering and the like of the Chinese cabbage MYB55gene are not reported.

Disclosure of Invention

In view of the above, one of the purposes of the present invention is to provide an application of overexpression Chinese cabbage MYB55 in improving the growth and development indexes of Brassica napus; the second purpose of the invention is to provide the application of the over-expression Chinese cabbage MYB55 in improving the yield of the cabbage type rape; the invention also aims to provide application of the over-expression Chinese cabbage MYB55 in improving lodging resistance of the cabbage type rape. The fourth purpose of the invention is to provide the application of the over-expression Chinese cabbage MYB55 in improving the resistance of cabbage type rape to sclerotinia rot.

In order to achieve the purpose, the invention provides the following technical scheme:

1. the application of the over-expression Chinese cabbage MYB55 in improving the growth and development indexes of the brassica napus is characterized in that: the amino acid sequence of the Chinese cabbage MYB55 is shown in SEQ ID No. 13.

Preferably, the full-length cDNA sequence of the Chinese cabbage MYB55 is shown in SEQ ID No. 11.

Preferably, the genomic sequence of the Chinese cabbage MYB55 is shown in SEQ ID No. 12.

Preferably, the improvement in growth and development index is at least one of root elongation and thickening, stem thickening or leaf thickening, flower number increase, bud number increase and pod number increase.

Preferably, the method for over-expressing the Chinese cabbage MYB55 comprises the steps of constructing a sense over-expression plant expression vector from a Chinese cabbage MYB55gene, then constructing a transformant to transform the cabbage type rape, and screening a transgenic plant to obtain the cabbage type rape with the over-expression Chinese cabbage MYB 55.

Preferably, the plant expression vector is obtained by inserting 151-1161bp shown in SEQ ID NO.11 between the CaMV35S promoter and the OCS terminator of the pFGC5941M vector.

Preferably, the transformant is agrobacterium tumefaciens LBA4404 containing the plant expression vector.

2. Application of overexpression Chinese cabbage MYB55 in improving yield of cabbage type rape.

3. Application of over-expression Chinese cabbage MYB55 in improving lodging resistance and/or sclerotinia resistance of cabbage type rape.

4. A method for obtaining cabbage type rape with high yield, lodging resistance, disease resistance and excellent growth and development comprises the following steps: inserting 151-1161bp shown in SEQ ID NO.11 between a CaMV35S promoter and an OCS terminator of a pFGC5941M vector to obtain a plant expression vector for over-expressing a cabbage MYB55, then transforming agrobacterium to obtain engineering bacteria, transforming the obtained engineering bacteria into a cabbage type rape host, and screening transgenic plants to obtain the cabbage type rape with high yield, lodging resistance, disease resistance and excellent growth and development.

The invention has the beneficial effects that: the invention provides the membership of MYB55gene in Chinese cabbage, its full-length cDNA sequence and genome sequence, coded protein characteristics, evolutionary relationship, expressed organ and tissue specificity, and confirms that BrMYB55gene expression is obviously up-regulated to improve the growth and development of cabbage type rape, for example, the xylem of stem and root is greatly thickened, stem and root are thicker, the number of primary branches is slightly increased, and the number of secondary branches is greatly increased; the pod angle number of a single plant is greatly increased, the yield of the single plant is greatly improved, and the yield is improved; the xylem of the stem of the transgenic plant becomes thicker, the bending resistance is improved, in addition, the root system becomes more developed, the lodging resistance of the transgenic plant is finally increased, in addition, sclerotinia is inoculated to the stem of the transgenic plant, and the disease spot is obviously smaller than that of a wild plant.

Therefore, the MYB55gene can greatly improve the overall growth and development and plant type of the cabbage type rape in agriculture, greatly improve lodging resistance, greatly improve resistance to sclerotinia which is a cancer disease that is not overcome by the rape so far, and have little side effect due to reverse change of characters, so the MYB55gene can be used for molecular breeding of the cabbage type rape.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is the electrophoresis diagram of the full-length cDNA and genome DNA amplification of Chinese cabbage BrMYB55gene family (A: full-length cDNA; B: genome DNA);

FIG. 2 shows the secondary structure of Chinese cabbage BrMYB55, the 4 segments from long to short in the longitudinal direction represent α helix, extension chain, β turn and random coil, respectively, and the numbers represent the number of amino acid residues in the protein.

FIG. 3 is the tertiary structure of the BrMYB55 protein.

FIG. 4 is a phylogenetic relationship of BrMYB55 with Arabidopsis and other species MYB transcription factors.

FIG. 5 shows the fluorescent quantitative RT-PCR analysis of MYB55gene family expression histological features.

FIG. 6 is a map of an overexpression vector for BrMYB 55.

FIG. 7 shows the tissue culture process of over-expressing BrMYB 55.

FIG. 8 shows the PCR identification of transgenic plants and their expression level comparison with that of control plants.

FIG. 9 shows phenotypic changes in growth and development of transgenic plants.

FIG. 10 shows phenotypic changes in transgenic plant yield.

FIG. 11 shows the histological anatomical changes of transgenic plants (A: WT plants; B: overexpression BrMYB55 transgenic plants; A, B: 500. mu.m).

FIG. 12 shows the research change of stalk bending strength of transgenic plants.

FIG. 13 shows the disease-resistant phenotypic changes of transgenic plants.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified, in the preferred examples are generally carried out according to conventional conditions, for example, as described in the molecular cloning protocols (third edition, J. SammBruk et al, Huangpetang et al, scientific Press, 2002), or according to the conditions recommended by the manufacturers.

The preferred embodiment uses plant material: the Chinese cabbage is selected from the spring Chinese cabbage of spectrum, which is provided by research center of rape engineering technology in Chongqing city and planted under conventional field test conditions. The seeds of the black-seed rape variety "Zhongshuang No. 10" were provided by Zhang Xun researchers at the institute of oil crops, academy of agricultural sciences, China.

Reagents and kits used in the preferred embodiments: the rapid extraction kit of the EASYspin plant RNA is purchased from Beijing Bomaide biotechnology limited; PrimeScript RT reagent Kit with gDNA Eraser (Perfect RealTime) reverse transcription Kit was purchased from TaKaRa, Dalian; the fluorescent quantitation kit FastStart Essential DNASGreen Master was purchased from Roche; various restriction enzymes XbaI, BamHI and SacI are products of Altao MBIFermestas company; murashige & Skoog (MS) medium was purchased from Duchefa, Netherlands; GoldView nucleic acid dyes were purchased from Baisheng Gene technologies, Inc., Beijing; CTAB, TE Buffer, DNA Loading Buffer, rifampicin (Rif), streptomycin (Str), kanamycin (Kan), ampicillin (Amp), cefotaxime sodium (Cef), agarose, tryptone, yeast extract, and asta herbicide were purchased from Shanghai Biotechnology services, Inc.;

the preferred embodiment employs the main instruments: ABI model 9700 PCR Instrument, Bio-Rad CFX96Touch, Applied Biosystems, USA^TMA fluorescent quantitative PCR instrument; and other conventional apparatus, equipment and facilities for molecular biology and plant genetic engineering.

Example 1 cloning of the Chinese cabbage MYB55Gene family

The primers used in this study are shown in Table 1.

TABLE 1 primers used in the examples of the present invention

(1) Extraction of Chinese cabbage genome total DNA and total RNA

Taking young leaves of Chinese cabbage plants, extracting total genomic DNA by a Cetyl Trimethyl Ammonium Bromide (CTAB) method, and evaluating the quality and concentration of a nucleic acid sample by a 1.0% agarose gel electrophoresis method and a spectrophotometry method. Meanwhile, the root, stem, leaf, flower, 30 days of seeds after flowering and 20 days of pod skin after flowering of Chinese cabbage are used as materials, column type small plant total RNA extraction kit is adopted to extract total RNA according to the instruction, DNase I is used to remove DNA impurities, and the total RNA is dissolved in pure water after ethanol precipitation. The nucleic acid sample is subjected to 1% agarose gel electrophoresis for quality detection, and the concentration and purity are measured by a Nanodrop ultraviolet spectrophotometer.

The electrophoresis result shows that the integrity of the total DNA of the Chinese cabbage genome extracted by the CTAB method is good, the RNA is completely digested, the purity detected by the spectrophotometry method is higher, and the DNA Polymerase Chain Reaction (PCR) amplification and Southern hybridization can be used. Electrophoretic analysis shows that the obtained total RNA characteristic band is clear, obvious RNA degradation and DNA pollution are avoided, the quality of spectrophotometry detection and evaluation is good, and the requirements of downstream experiments can be met.

(2) Obtaining the first chain of total cDNA of each organ of Chinese cabbage

Equally mixing the RNAs extracted in the step (1), and performing reverse transcription by using a PrimeScript RT reagentKit with gDNAeraser (Perfect read Time) of Takara company to obtain a first chain of the total cDNA of each organ of the Chinese cabbage.

(3) Electronic cloning of Chinese cabbage MYB55gene family

The Arabidopsis MYB55gene (AtMYB55) is located on chromosome 4 (AT4G01680), and the cDNA (AT4G01680) is 1248 bp. The cDNA sequence is submitted to NCBI (http:// www.ncbi.nlm.nih.gov/BLAST /) website for nucleic acid sequence alignment (nucleotid BLAST), Brassica rapa (tail: 3711) is input into Organism, other and Reference RNA sequences (refseq-RNA) are selected from Database, and Somewhar similar sequences (blatsn) are selected for optimization, and then BLAST is clicked for alignment, and the alignment result shows that the gene annotated as BrMYB55 is not present in GenBank, but the gene homology scores of other MYB transcription factors with 1 annotation are higher, and the created sequence is aligned to an Arabidopsis website to be found to be a vertical homologous gene of Arabidopsis thaliana AtMYB55, so that only 1 MYB55gene may exist in cabbage presumably and the corresponding independent gene is named as BrMYB 55.

(4) Cloning of full-length cDNA of Chinese cabbage MYB55gene family

The first strand of the total cDNA of the Chinese cabbage mixed organ is used as a template, the primer combination listed in the table 1 is used for PCR amplification, and agarose gel electrophoresis of the PCR amplification product shows that a band of about 1400bp is amplified by the primer combination FBrMYB55+ RBrMYB55 (figure 1, A). After glue recovery and TA cloning, PCR detection of bacterial liquid of batch clones shows that the length of an inserted fragment has polymorphism, the polymorphism is sent to sequencing, BLASTn of a sequencing result and comparison with AtMYB55 show that the fragment is a Chinese cabbage MYB55gene, the length of standard full-length cDNA obtained by sequencing a band is 1394bp, and the standard full-length cDNA is named BrMYB55mRNA as shown in SEQ ID NO. 11.

(5) Cloning of Chinese cabbage MYB55gene family genome DNA

The template is replaced by the total DNA of the Chinese cabbage genome, PCR amplification is carried out as before, and the electrophoresis result shows that a band of about 1600bp is amplified by the primer combination FBrMYB55+ RBrMYB55 (figure 1, B). After the recovery of the gel and the cloning of TA, the PCR detection of the bacterial liquid has no polymorphism, the accurate length obtained by sequencing is 1588bp, which is named as BrMYB55gene and is shown as SEQ ID NO. 12.

Example 2 bioinformatic analysis of the BrMYB55Gene family

Sequence alignment, Open Reading Frame (ORF) search and translation were performed on Vector NTI Advance 11.5, BLAST and CDD search of protein sequences were performed on the http:// www.ncbi.nlm.nih.gov/website, protein structure analysis was performed on bioinformatics websites providing links to the http:// bip. weizmann. ac. il/and www.expasy.org, and gene and protein sequence multiple alignment and clustering were performed on the http:// products. toulouse. in. fr/multalin. html and http:// www.ebi.ac.uk/clustalw/etc.

1. Structural analysis of Chinese cabbage MYB55gene family

1.1 structural parameters of the Chinese cabbage MYB55gene family

The DNA sequence of the BrMYB55gene is 1588bp, 3 exons and 2 introns exist, the longest standard mRNA is 1394bp, the longest 5 ' non-coding region (5 ' UTR) is 141bp, the longest 3 ' UTR is 242bp, the coding region (open reading frame ORF including stop codon) is 1011bp, the GC content of the coding region is 43.32%, the GC content is obviously higher than that (28.3%) of the non-coding region (5 ' UTR +3 ' UTR + intron), and the structural characteristics of the functional gene (Table 2) are met.

TABLE 2 basic characteristics of the genes of the BrMYB55 family

1.2 basic parameters, subcellular localization and possible post-transcriptional modifications of the Chinese cabbage MYB55 family proteins

The deduced BrMYB55 protein is 336 amino acid residues, has the molecular weight of 37.72kD, has the isoelectric points of 6.9, contains 31.77 percent of charged amino acid, 10.41 percent of acidic amino acid, 11.38 percent of basic amino acid, 37.76 percent of polar amino acid and 26.71 percent of hydrophobic amino acid, is a basic protein, and contains the highest content of leucine, threonine and asparagine in single amino acid.

SignalP5.0 predicted them to have no signal peptide. Softberry-ProComp, WoLFPSORT and Plant-mPoloc predicted that they localized to the nucleus; TMpred predicts that they do not have any transmembrane domains. NetPhos 3.1 predicts that they have 48 potential phosphorylation sites, mainly serine phosphorylation sites (table 3).

TABLE 3 basic characteristics of the proteins encoded by the BrMYB55 family

	BrMYB55		BrMYB55
				Number of amino acid residues (aa)	336	3 kinds of the most abundant amino acids	L,T,N
Molecular weight (kDa)	37.72	Number of serine phosphorylation sites	26
				Isoelectric point	6.9	Number of threonine phosphorylation sites	19
Charge (pH 7)	-0.3	Number of tyrosine phosphorylation sites	3
				Charged amino acid proportion (%)	31.77	Conserved domains	SANT
Acid amino acid proportion (%)	10.41	α -helix (%)	26.49
				Basic amino acid proportion (%)	11.38	Extension chain (%)	15.48
Polar amino acid proportion (%)	37.76	β -Angle (%)	8.93
				Hydrophobic amino acid ratio (%)	26.71	Random crimp (%)	49.11

1.3 conserved domain, conserved motif and higher-order structure of Chinese cabbage MYB55 family protein

NCBI Conserved Domain Search (NCBI Conserved Domain Search) indicated that the SANT Conserved Domain (cl28544), the DNA binding Domain of the transcription factors SWI3, ADA2, N-CoR and TFIIIB, was found in the 1 protein of the BrMYB55 family.

The SOPMA software predicted that the secondary structure of BrMYB55 was predominantly random coil (49.11%), followed by α -helix (26.49%) and extension strand (15.5%), β -turn (8.9%) (table 3, fig. 2).

Swiss-Model predicted their full three-dimensional structure (FIG. 3).

2. Homology and phylogenetic relationship of Chinese cabbage MYB55 family

2.1 homology of nucleic acids to protein levels

Pairwise alignment on Vector NTI showed that the homology of the full-length gene of BrMYB55 with AtMYB55 was highest, the consensus rate at gDNA level calculated by Vector NTI was 72.1%, the consensus rate at coding region level was 83.2%, and the consensus rate and similarity rate at coding protein level were 84.6% and 86.6%, respectively (table 4). At the nucleic acid level, they share some homology with some other MYB genes. They are significantly more conserved in coding regions than in non-coding regions, but introns are conserved at splice boundaries, and there are also some conserved local regions in the 5 'UTR and 3' UTR that should be related to gene expression activities. At the protein level, they also share some homology with many other MYB proteins.

TABLE 4 genomic sequence identity (italic, top), coding region identity (italic, bottom), protein identity (normal, top) and protein similarity (normal, bottom) (%) between the BrMYB55gene and the AtMYB55 gene

2.2 phylogenetic relationships

Arabidopsis MYB protein, shown by BLASTp to have similarity to cabbage MYB55, was downloaded and used together with cabbage MYB55 protein to construct a phylogenetic tree on Vector NTI Advance using ClustalW multiple alignments (FIG. 4). The phylogenetic tree reveals that the Chinese cabbage MYB55 protein and the Arabidopsis MYB55 are firstly gathered into one type, and the fact that the Chinese cabbage MYB55 protein is a vertical homologous gene (orthologs) of AtMYB55 is shown.

Example 3 relationship between expression characteristics of Chinese cabbage MYB55 family and sclerotinia sclerotiorum resistance

Selecting roots, stems, leaves, flowers of Chinese cabbage materials, seeds 30 days after the flowers are bloomed, 6 tissue organs of pod skins 20 days after the flowers are bloomed, and RNA samples of mature leaves 2cm around lesion spots after 0h, 0.5h, 3h, 9h, 24h and 48h of sclerotinia are inoculated as templates, carrying out reverse transcription by using PrimeScript RT reagent Kit with gDNA Eraser to obtain total cDNA, and analyzing the expression histological characteristics of MYB55gene families by using fluorescence quantitative RT-PCR. The expression level of BrMYB55 was determined using the primer combination FBrMYB55Q + RBrMYB55Q, and the reactions were all performed in a 25. mu.l standard FastStart Essential DNA Green Master-PCR system, and the procedures of the PCR reactions were as follows: pre-denaturation at 95 ℃ for 10min → 45 amplification cycles (denaturation at 95 ℃ for 10s → annealing at 62 ℃ for 30s) → melting curve 65 ℃ to 95 ℃.

RT-PCR results show that BrMYB55 is expressed in the above organs, but is significantly expressed in stems, and the expression level is far higher than that of other organs. In addition, BrMYB55 was rapidly induced by sclerotinia sclerotiorum and was rapidly upregulated 0.5h after inoculation with sclerotinia sclerotiorum. Indicating that BrMYB55 participates in the sclerotinia sclerotiorum resisting process of rape (figure 5).

Example 4 application of Chinese cabbage MYB55gene family

1. Cloning of sense fragment of BrMYB55gene family member

The method comprises the steps of adopting Chinese cabbage mixed total cDNA as a template, adopting a primer combination FBMYB55OE + RBMYB55OE to amplify a sense fragment (BrMYB55ox) of a BrMYB55gene, wherein the size of a product fragment shown by electrophoresis is the same as that of an expected fragment, respectively recovering the fragment, connecting the fragment with pMD19-T, transforming DH5 α, selecting PCR positive monoclonal bacteria liquid for sequencing, and displaying that the length of BrMYB55ox is 1011bp, and compared with the full-length cDNA sequence of the gene, the cDNA sequence does not contain 5' UTR, has the same interval and does not have mutation.

2. Construction of sense transformation plant expression vector for BrMYB55gene family member

The gene fragment is subjected to partial enzyme digestion in a short time and accurately to minutes by using trace NcoI (ice water bath is adopted for stopping digestion in the middle, six bands of 1019/1029, 919, 843, 743, 182 and 106bp coexist in equal quantity during electrophoresis to form an optimal state, the 1019bp fragment is lost or is in a small quantity to indicate failure, the enzyme quantity of the NcoI needs to be further reduced and the enzyme digestion time needs to be shortened), a 1019bp complete gene fragment band is recovered for later use (other bands are incomplete gene fragments), a pFGC5941M is subjected to complete enzyme digestion by adopting NcoI + XbaI to recover a skeleton for later use, sticky end connection is carried out between the gene and the skeleton of the vector, the target gene is subcloned between a CaMV35S promoter and an OCS 3' in the pFGC59 5941M, a pFGC 5941M-BrB55ox vector is formed, the gene is transformed into the pFGC5 α, Kan-resistant clone is obtained, and the plasmid is subjected to PCR extraction by using each primer combination to obtain a PCR (4404 gram positive plasmid).

3. Agrobacterium-mediated sense excess plant expression vector pFGC5941M-BrMYB55ox for transforming black-seeded brassica napus

All tissue culture operations are carried out under standard plant tissue culture conditions, the cleanliness grades of the superclean bench, the culture room and the domestication room are respectively 100 grades, 10000 grades and 100000 grades, and corresponding reagents, materials and vessels are subjected to aseptic treatment according to regulations. Soaking seeds No. 10 of a typical black-seed variety of the cabbage type rape in clear water for 1-2 h, disinfecting the surfaces of the seeds with 75% ethanol for 1min, washing the seeds with sterile water for 3 times, soaking the seeds with 0.1% mercuric chloride for 15min, washing the seeds with the sterile water for multiple times, and then inoculating the seeds to an MS solid culture medium (MS powder is 4.41g/L, Phytagel is 2.6g/L, cane sugar is 30.0mg/L, pH is 5.8, and sterilizing the seeds by heat sterilization at a pot temperature; the culture medium is a liquid culture medium without adding any Phytagel, and the culture is carried out at 25 ℃ under 2000Lux illumination for 16h/d photoperiod (the culture conditions in the later tissue culture rooms are the same except for specially noted ones). The hypocotyl of the aseptic seedling with the seedling age of about 8 days is cut into small sections with the length of about 0.5-1.0 cm, and the small sections are inoculated to a pre-culture medium MSp (MS culture medium +1.0 mg/L6-benzylaminopurine (6-BA) +1.0mg/L2, 4-dichlorophenoxyacetic acid (2, 4-D)) for pre-culture for 3 days.

The engineering strain preserved at minus 80 ℃ is added with 100.0mg/L Kan +20.0mg/L Str +40.0mg/L Rif in LB liquid culture medium, and is subjected to shaking culture at 250r/min at 28 ℃ for 1-2 days, so that the agrobacterium is grown to logarithmic phase, and is subjected to transfer culture once. Centrifuging at room temperature at 5000rpm for 10min to collect thallus, and treating with staining culture medium MSm [ MS liquid culture medium +1.0mg/L2, 4-dichlorophenoxyacetic acid (2,4-D) +1.0 mg/L6-benzylaminopurine (6-BA) +100 μ M Acetosyringone (AS)]Adjusting bacterial concentration to OD₆₀₀About 0.5, namely the dip dyeing solution.

Will preImmersing the cultured hypocotyl segment in the staining solution for 5-10min while intermittently and gently shaking, sucking off the redundant bacteria liquid from the hypocotyl segment on sterilized paper, and inoculating to co-culture medium MSc (MS solid medium +1.0 mg/L6-BA +1.0mg/L2,4-D +50 μ M AS)]In the medium, the cells were cultured at 23.5 ℃ for 48 hours in the dark. Sterilizing liquid culture medium MSk (MS liquid culture medium +1.0mg/L2,4-D +1.0 mg/L6-BA +500mg/L cephamycin (Cef)]Soaking and washing explant for 3 × 10min, blotting surface liquid with sterilized paper, transferring to induction screening culture medium MSi [ MS solid culture medium +1.0 mg/L6-BA +1.0mg/L2,4-D +500mg/L Cef +15ppm Basta]Culturing for 1 time in medium culture for about 2 weeks, subculturing for 1 time until macroscopic resistant callus grows out, and transferring to differentiation culture medium MSd [ MS solid culture medium +4.0 mg/L6-BA +2.0mg/L Zeatin (ZT) +5.0mg/L AgNO₃+500mg/L Cef+15ppm Basta]Culturing for more than 14 days, inducing callus to differentiate into small bud, culturing in stem differentiation culture medium MSs (MS solid culture medium +3.0 mg/L6-BA +2.0mg/L ZT +500mg/L Cef +10ppm Basta) to grow small stem, culturing in long stem culture medium MSe (M solid culture medium +0.05 mg/L6-BA +500mg/L Cef +10ppm Basta) to grow complete stem slice, and culturing in rooting culture medium MSr [ MS solid culture medium +2mg/L naphthylacetic acid (NAA)]Culturing until developed root system grows, domesticating rooted plantlets, transplanting into a pot containing a mixture of sterilized perlite, vermiculite and turfy soil (mass ratio is 1:1:1), and managing according to greenhouse pot culture to finally obtain multiple regenerated plants (figure 7).

Meanwhile, the medium-double No. 10 regeneration plants obtained under the same tissue culture condition without Basta screening pressure are used as non-transgenic negative controls.

4. Identification of transgenic plants

(1) Basta reexamination identification of transgenic plants

The leaves of the transgenic regenerated plants and the control plants were stained with a Basta solution at a concentration of 200 ppm.

(2) PCR identification of transgenic plants

And (3) respectively taking leaves of the transgenic regeneration plant and the negative control plant, extracting total DNA of the genome, and performing composite PCR detection by adopting 2 pairs of primer combinations of FBMYB55OE + ROCST5N and F35S3N + RBMYB55 OE. The positive transgenic regenerated plant can amplify a band with the same size as the positive control (CK + and engineering strain) under the detection of 2 primer combinations, and the negative control plant (WT) has no band (figure 8).

5. Investigation of transgenic plant traits

Transgenic positive plants develop obviously later than control wild plants, and the height of the plants is obviously lower than that of the control plants before full-bloom stage. However, the lateral growth of the transgenic plants was significantly more advanced than that of the control plants, and was manifested on the vegetative organs of the transgenic plants, such as the lengthened and thickened roots, the thickened stems, and the enlarged leaves (FIG. 9). The reproductive growth of the transgenic plants was significantly delayed by 3-5 days compared to the control wild-type plants, but the number of flowers, buds, and siliques, etc., of the transgenic plants were not decreased, but increased, and the yield of the whole transgenic plants was increased (FIG. 10). The tissue anatomy study found that the stem vascular bundle of the transgenic plant became larger, the xylem became thicker and its area became larger (FIG. 11). And the stalk resistance of the rape at the harvest stage is analyzed, and the stalk resistance of the transgenic plant is greatly improved (figure 12), and the stalk resistance is an important index suitable for the lodging resistance of the small-scale transgenic plant, which indicates that the BrMYB55 participates in the lodging resistance process of the rape. In addition, the leaf plaque area of the leaf is counted after the contrast and transgenic plants in the 9-12 leaf period are inoculated with sclerotinia sclerotiorum in vitro for 24 hours, and 3 or 4 true leaves are poured from the transgenic plants. According to statistical results, leaf plaque area was found to be significantly reduced in BrMYB55ox transgenic plants (fig. 13). The results show that BrMYB55 participates in the growth and development of plants and has the function of improving the growth and development, and BrMYB55 also participates in and positively regulates the anti-lodging and anti-sclerotinia sclerotiorum processes of rape.

6. Further description of the application forms

1. The genes and fragments thereof in the present invention, in addition to the nucleotide sequences listed in the sequence listing, also include sequences from other MYB55 alleles from cabbage, and also MYB55gene sequences from other subspecies, ecotypes or varieties of cabbage, although they may differ slightly from the nucleotide sequences listed in the sequence listing.

2. The gene and its fragment of the present invention include any nucleotide sequence having more than 98.00% identity to the nucleotide sequence listed in the sequence listing, except for the nucleotide sequence listed in the sequence listing, which is continuous with the nucleotide sequence of 80bp or more.

3. In the overexpression vector constructed in the present invention, similar effects can be obtained by transforming any one of vectors except for the transformation of pFGC5941M-BrMYB55ox as exemplified in the preferred embodiment.

4. The genes and fragments thereof of the present invention can be applied to other species besides Brassica napus as exemplified in the preferred embodiment.

5. The gene and the fragment thereof in the invention can adopt the technologies of antisense, RNA interference, CRES-T and the like to inhibit the expression of MYB55gene in addition to the characteristics of improving the growth and development, increasing the yield, resisting diseases and the like of plants by adopting the sense over-expression technology as shown in the prior embodiment, thereby influencing the characteristics of the growth and development, reducing the yield, lodging, diseases and the like of the plants.

6. The gene and its fragment of the present invention can be constructed by using other vectors in addition to the vector construction by pFGC5941M as exemplified in the preferred embodiment; the vector constructs of the present invention can be used for plant transformation by methods other than the Agrobacterium tumefaciens LBA 4404-mediated modified leaf disc method as exemplified in the preferred embodiment.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

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<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

cttccatggt taatccccaa cta 23

<210>5

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

gatttctgcc cagtgctctg aa 22

<210>6

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

tctgccaagc ccgttccctt 20

<210>7

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

ccatgggaag acattcatgc tgttac 26

<210>8

<211>31

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

tctagattaa atatggccat atgcatgttg c 31

<210>9

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

ggaagttcat ttcatttgga gag 23

<210>10

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gctcaggttt tttacaacgt gcac 24

<210>11

<211>1394

<212>DNA

<213> cabbage (Brassica rapa)

<400>11

atcttctcac ttcaaactct ctctatctct ctctcacctc atcttaagat ttctctgaaa 60

gctgactaca aagccttttc taaaaacaaa caagacgatc cttctctaat tgatatttta 120

tttatatata aaattttaaa aatgggaaga cattcatgct gttacaaaca gaagctgagg 180

aaaggacttt ggtctcctga agaagacgag aagcttctta ggtacatcac taagtacggc 240

catggctgct ggagctctgt ccctaaacaa gctggtttgc agagatgtgg aaagagctgt 300

agattaagat ggataaacta tctaagacca gatttgaagc gaggagcatt ttcacaggat 360

gaagaaaacc ttattattga acttcatgcc gttcttggca acaggtggtc tcagattgct 420

gcacagcttc ctggtagaac cgacaatgaa atcaagaatc tatggaactc ttccttaaag 480

aagaaactga ggctgagagg aattgatccg gttacacaca agctcttaac cgaaattgaa 540

accggtacag atgacaatac cacaccggtt gagaagtgtc aaacgaccta cctcattgag 600

acagaaggct cctctagtac caccactggc agtactaacc acaacaacag caacaccgat 660

catctttata ccggaaattt tggtttccaa cggttaagtc ttgagactgg ttcaagaata 720

caaaccggca tctggattcc ccaaaccggg agaaatcatc atgttgatac cgtacctagt 780

gcagtggtgc tacccggttc aatgttctca tctggcttaa ccgattcaac aaccggttac 840

agatcatcca atctcggttt aattgaattg gataactcat tctcgaccgg gccaatggtt 900

acagagcagc aacttcaaga gagtaactac aacaattcga cattctttgg aactgggaat 960

ctgaattggg gattaaccat ggaagaaaat caatttacaa tatcgaataa ttcgttacag 1020

aatcactcaa actcgtcgtt gtatagtgaa atcaagtccg agaccaattt tttcggtacg 1080

gaggctgcaa atgttggtat gtggccatgt aaccagctcc agcctcagca acatgcatat 1140

ggccatattt aaaatcttct tgtatattat aaggtgtgtg gatcttcttt ttcttcttca 1200

agttattttt ctttattcca aatatcgagt tttatttata atggtttgtg tatataaagt 1260

ttgtgttata ttcatttaat ctaagggtgt tgttcttaca ttttctttta tttgatgtac 1320

tttgtgaagc atagttttct ggactttgag attttgtttg tctattcttg ctggatcaat 1380

gacatttatt tctt 1394

<210>12

<211>1588

<212>DNA

<213> cabbage (Brassica rapa)

<400>12

atcttctcac ttcaaactct ctctatctct ctctcacctc atcttaagat ttctctgaaa 60

gctgactaca aagccttttc taaaaacaaa caagacgatc cttctctaat tgatatttta 120

tttatatata aaattttaaa aatgggaaga cattcatgct gttacaaaca gaagctgagg 180

aaaggacttt ggtctcctga agaagacgag aagcttctta ggtacatcac taagtacggc 240

catggctgct ggagctctgt ccctaaacaa gctggtaatt taatttctct ttgctttcat 300

ccaattatta ctttgttgtg cttgattaaa tactccttcc taattcttga attatcattt 360

ttttattaat tttttttagg tttgcagaga tgtggaaaga gctgtagatt aagatggata 420

aactatctaa gaccagattt gaagcgagga gcattttcac aggatgaaga aaaccttatt 480

attgaacttc atgccgttct tggcaacagg taaagaaccc cgtgctgttt ctctgatcct 540

tatgcggcat gtaagtgtat ttatatatgt aacccatgtt gctttggtct tggtttaggt 600

ggtctcagat tgctgcacag cttcctggta gaaccgacaa tgaaatcaag aatctatgga 660

actcttcctt aaagaagaaa ctgaggctga gaggaattga tccggttaca cacaagctct 720

taaccgaaat tgaaaccggt acagatgaca ataccacacc ggttgagaag tgtcaaacga 780

cctacctcat tgagacagaa ggctcctcta gtaccaccac tggcagtact aaccacaaca 840

acagcaacac cgatcatctt tataccggaa attttggttt ccaacggtta agtcttgaga 900

ctggttcaag aatacaaacc ggcatctgga ttccccaaac cgggagaaat catcatgttg 960

ataccgtacc tagtgcagtg gtgctacccg gttcaatgtt ctcatctggc ttaaccgatt 1020

caacaaccgg ttacagatca tccaatctcg gtttaattga attggataac tcattctcga 1080

ccgggccaat ggttacagag cagcaacttc aagagagtaa ctacaacaat tcgacattct 1140

ttggaactgg gaatctgaat tggggattaa ccatggaaga aaatcaattt acaatatcga 1200

ataattcgtt acagaatcac tcaaactcgt cgttgtatag tgaaatcaag tccgagacca 1260

attttttcgg tacggaggct gcaaatgttg gtatgtggcc atgtaaccag ctccagcctc 1320

agcaacatgc atatggccat atttaaaatc ttcttgtata ttataaggtg tgtggatctt 1380

ctttttcttc ttcaagttat ttttctttat tccaaatatc gagttttatt tataatggtt 1440

tgtgtatata aagtttgtgt tatattcatt taatctaagg gtgttgttct tacattttct 1500

tttatttgat gtactttgtg aagcatagtt ttctggactt tgagattttg tttgtctatt 1560

cttgctggat caatgacatt tatttctt 1588

<210>13

<211>336

<212>PRT

<213> cabbage (Brassica rapa)

<400>13

Met Gly Arg His Ser Cys Cys Tyr Lys Gln Lys Leu Arg Lys Gly Leu

1 5 10 15

Trp Ser Pro Glu Glu Asp Glu Lys Leu Leu Arg Tyr Ile Thr Lys Tyr

20 25 30

Gly His Gly Cys Trp Ser Ser Val Pro Lys Gln Ala Gly Leu Gln Arg

35 40 45

Cys Gly Lys Ser Cys Arg Leu Arg Trp Ile Asn Tyr Leu Arg Pro Asp

50 55 60

Leu Lys Arg Gly Ala Phe Ser Gln Asp Glu Glu Asn Leu Ile Ile Glu

65 70 75 80

Leu His Ala Val Leu Gly Asn Arg Trp Ser Gln Ile Ala Ala Gln Leu

85 90 95

Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Leu Trp Asn Ser Ser Leu

100 105 110

Lys Lys Lys Leu Arg Leu Arg Gly Ile Asp Pro Val Thr His Lys Leu

115 120 125

Leu Thr Glu Ile Glu Thr Gly Thr Asp Asp Asn Thr Thr Pro Val Glu

130 135 140

Lys Cys Gln Thr Thr Tyr Leu Ile Glu Thr Glu Gly Ser Ser Ser Thr

145 150 155 160

Thr Thr Gly Ser Thr Asn His Asn Asn Ser Asn Thr Asp His Leu Tyr

165 170 175

Thr Gly Asn Phe Gly Phe Gln Arg Leu Ser Leu Glu Thr Gly Ser Arg

180 185 190

Ile Gln Thr Gly Ile Trp Ile Pro Gln Thr Gly Arg Asn His His Val

195 200 205

Asp Thr Val Pro Ser Ala Val Val Leu Pro Gly Ser Met Phe Ser Ser

210 215 220

Gly Leu Thr Asp Ser Thr Thr Gly Tyr Arg Ser Ser Asn Leu Gly Leu

225 230 235 240

Ile Glu Leu Asp Asn Ser Phe Ser Thr Gly Pro Met Val Thr Glu Gln

245 250 255

Gln Leu Gln Glu Ser Asn Tyr Asn Asn Ser Thr Phe Phe Gly Thr Gly

260 265 270

Asn Leu Asn Trp Gly Leu Thr Met Glu Glu Asn Gln Phe Thr Ile Ser

275 280 285

Asn Asn Ser Leu Gln Asn His Ser Asn Ser Ser Leu Tyr Ser Glu Ile

290 295 300

Lys Ser Glu Thr Asn Phe Phe Gly Thr Glu Ala Ala Asn Val Gly Met

305 310 315 320

Trp Pro Cys Asn Gln Leu Gln Pro Gln Gln His Ala Tyr Gly His Ile

325 330 335

Claims (9)

1. Over-expression Chinese cabbageMYB55The application of the compound preparation in improving the growth and development indexes of the brassica napus is characterized in that: the Chinese cabbageMYB55The amino acid sequence of (A) is shown as SEQ ID No. 13; the index for improving growth and development is at least one of root lengthening and thickening, stem thickening or leaf thickening, flower number increasing, bud number increasing and pod number increasing.

2. Use according to claim 1, characterized in that: the Chinese cabbageMYB55The mRNA sequence of (A) is shown as SEQ ID No. 11.

3. Use according to claim 1, characterized in that: the Chinese cabbageMYB55The genome sequence of (A) is shown in SEQ ID No. 12.

4. Use according to claim 1, characterized in that: said overexpression Chinese cabbageMYB55The method comprises mixing Chinese cabbageMYB55Constructing a plant expression vector with positive sense over-expression by gene, constructing a transformant to transform the cabbage type rape, and screening transgenic plantsObtaining the over-expression Chinese cabbageMYB55The brassica napus.

5. Use according to claim 4, characterized in that: the plant expression vector is obtained by inserting 151-1161bp shown in SEQ ID NO.11 between a CaMV35S promoter and an OCS terminator of a pFGC5941M vector.

6. Use according to claim 4, characterized in that: the transformant is agrobacterium tumefaciens LBA4404 containing the plant expression vector.

7. Over-expression Chinese cabbageMYB55The application of the method in improving the yield of the cabbage type rape is characterized in that: the Chinese cabbageMYB55The amino acid sequence of (A) is shown in SEQ ID No. 13.

8. Over-expression Chinese cabbageMYB55The application of the compound in improving the lodging resistance and/or sclerotinia resistance of the cabbage type rape is characterized in that: the Chinese cabbageMYB55The amino acid sequence of (A) is shown in SEQ ID No. 13.

9. A method for obtaining cabbage type rape with high yield, lodging resistance, disease resistance and excellent growth and development is characterized by comprising the following steps: inserting 151-1161bp shown in SEQ ID NO.11 between CaMV35S promoter and OCS terminator of pFGC5941M vector to obtain over-expression Chinese cabbageMYB55Then transforming agrobacterium to obtain engineering bacteria, transforming the obtained engineering bacteria into a cabbage type rape host, and screening transgenic plants to obtain the cabbage type rape with high yield, lodging resistance, disease resistance and excellent growth and development; the excellent growth development is at least one of root lengthening and thickening, stem thickening or leaf thickening, flower number increasing, bud number increasing and pod number increasing.

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