CN112626259A - Method for identifying sclerotinia rot resistant rape strain and application - Google Patents
Method for identifying sclerotinia rot resistant rape strain and application Download PDFInfo
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- CN112626259A CN112626259A CN202110044741.XA CN202110044741A CN112626259A CN 112626259 A CN112626259 A CN 112626259A CN 202110044741 A CN202110044741 A CN 202110044741A CN 112626259 A CN112626259 A CN 112626259A
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Abstract
The invention belongs to the technical field of rape breeding, and particularly relates to a method for identifying an sclerotinia rot resistant rape line and application thereof. The BnUGT74B1 gene related by the invention is cloned from rape, and the expression level of the gene in different non-transgenic materials is analyzed, so that the rape line with stronger sclerotinia sclerotiorum disease resistance can be identified according to the difference of the expression level of the gene, thereby improving the sclerotinia sclerotiorum disease resistance of the rape by using the non-transgenic materials and providing new genetic resources for breeding.
Description
Technical Field
The invention belongs to the technical field of rape breeding, and particularly relates to a method for identifying an sclerotinia rot resistant rape line and application thereof.
Background
The sclerotinia rot of rape is the most main disease in the main rape producing area in China, and leads to the rape yield loss of 10-20% all the year round. Sclerotinia sclerotiorum is a fungal disease caused by infection of Sclerotinia sclerotiorum (Lib.) de bark, the pathogenic bacteria are typical dead body nutritional pathogenic bacteria, chemical control often influences the use effect due to inaccurate control of the disease onset period of the Sclerotinia sclerotiorum and improper control of the use method, and a disease-resistant rape variety is urgently needed in production. However, no germplasm resource for immunizing sclerotinia sclerotiorum is found in rape and related species at present, and meanwhile, because the identification procedure of sclerotinia sclerotiorum is complicated and the workload is great, it is very difficult to obtain resistance resources in natural resources. New identification methods and techniques are therefore needed.
Thioglucoside (hereinafter abbreviated as thioglucoside) is an important secondary metabolite containing sulfur and nitrogen, and mainly exists in plants of Cappariales, such as Chinese cabbage, caulis et folium Brassicae Capitatae, caulis et folium Brassicae campestris, radix Raphani, caulis et folium Brassicae Junceae, horse radish, and Arabidopsis thaliana. The glucosinolate has important effects on the aspects of plant growth and development, defense reaction and the like.
Glucosinolates play an important role in the growth and development of plants of the order Capillales. The glucosinolate and the degradation product thereof participate in the defense reaction of plants, effectively resist the influence of plant diseases and insect pests on the plants, and are mainly expressed as follows: the aliphatic glucosinolate can resist the invasion of the leaf budworm and some host pathogenic bacteria; the indole glucosinolates are effective against insect oviposition and non-host pathogenic bacteria. The biological activity of thioglycoside, especially in plant defense, is mainly functional through its degradation products. When plant tissues meet the stresses of various organisms and non-organisms such as chewing and eating of insects or invasion of pathogenic bacteria, the synthesis or degradation and metabolism of thioglycoside can be induced, so that isothiocyanate, thiocyanate, nitrile compounds, important sulfides and the like are generated, and a certain defense effect on diseases and insect pests is achieved.
The defense mode of plants against antigen invasion is divided into a constitutive type and an inducible type, the disease-resistant type of glucosinolate belongs to the former type, and the synthesis of the glucosinolate in plant tissues can still exist even if the plant tissues are not induced or stressed by the outside. When pathogenic bacteria invade plant tissues and destroy organelles for isolating the myrosinase and the glucosinolate, the myrosinase and the glucosinolate are connected to catalyze the degradation of the glucosinolate, and degradation products of the glucosinolate further play a defense function, so that the aims of reducing the diffusion of the pathogenic bacteria and even killing the pathogenic bacteria are fulfilled.
Pathogenic bacteria are classified into two major groups, namely, a Biotrophic pathogen (Biotrophic pathogen) and a Necrotrophic pathogen (Necrotrophic pathogen) according to their nutritional patterns, wherein a Biotrophic pathogen, which is called powdery mildew (Erysiphe cichororaceae), Albugo (Albugo Candida), etc., if it absorbs nutrients from host cell tissues and does not cause death of host cells soon; if the pathogenic bacteria kill host cells or tissues first, then the pathogenic bacteria can absorb nutrients from the dead cells and propagate further, the pathogenic bacteria are called dead body nutritional pathogenic bacteria, such as Sclerotinia sclerotiorum (sclerotiorum) and the like. The living pathogenic bacteria can not damage the cell or tissue structure of the host at the beginning, and the glucosinolate can not be degraded, so the glucosinolate can not be induced by the living nutritional pathogenic bacteria rapidly under the general condition, and the pathogenic bacteria can also become very sensitive to the degradation products of the glucosinolate; the dead body nutritional pathogenic bacteria directly destroy the cell or tissue structure of the plant to cause the degradation of the glucosinolate, and the glucosinolate degradation products are connected for a long time to ensure that the glucosinolate degradation products have the capability of resisting the toxicity of the glucosinolate degradation products. Therefore, the disease resistance of the plants can be effectively influenced by changing the glucosinolate component and the content of the plants. It has been reported that indoxyl glucosinolates, particularly 3-indolylmethylglucosinolates, found in seeds are significantly associated with sclerotinia rot of Brassica napus (Leyunchang, 2001; Zhao and Meng, 2003).
Glycosyltransferases are a multi-member family of genes that are widely present in all organisms and catalyze glycosyl transfer, termed uridine diphosphate glycosyltransferases (UGT) because their glycosyl donor is uridine diphosphate glucuronic acid (UDP-glycosyltransferases) (machi chen et al, 2013). UGT plays an important role in regulating plant hormone balance, defense against detoxification, and modification of secondary metabolites, with UGT74B1 playing a key role in plant synthesis of indole glucosinolates (Grubb et al, 2004). An enzyme encoded by arabidopsis UGT74B1 glycosylates thioacetaldehyde oxime acid generated at the upstream in an indole thioglycoside synthesis pathway to form desulfthioside, and then the thioside is catalyzed by sulfotransferase to form complete thioglycoside (Wangjiangsheng et al, 2012).
Disclosure of Invention
The invention provides a method for identifying an sclerotinia sclerotiorum line and application thereof aiming at solving part of problems in the prior art or at least alleviating part of problems in the prior art. The invention utilizes the gene BnUGT74B1 to establish the relationship between gene expression quantity and disease resistance, and identifies new disease-resistant resources according to the gene expression quantity in a specific period, thereby improving the sclerotinia rot resistance of rape.
The invention is realized in such a way, and the method for identifying the sclerotinia sclerotiorum strain comprises the step of detecting the expression level of the BnUGT74B1 gene after the sclerotinia sclerotiorum is inoculated at the late flowering phase of the rape, wherein the resistance line with the remarkably improved expression level of the BnUGT74B1 gene is adopted.
The invention also discloses application of the method for identifying the sclerotinia rot resistant rape line in the breeding of the excellent rape variety.
The BnUGT74B1 gene related by the invention is cloned from rape, and the expression level of the gene in different non-transgenic materials is analyzed, so that the rape line with stronger sclerotinia sclerotiorum disease resistance can be identified according to the difference of the expression level of the gene, thereby improving the sclerotinia sclerotiorum disease resistance of the rape by using the non-transgenic materials and providing new genetic resources for breeding.
In summary, the advantages and positive effects of the invention are:
according to the invention, the expression level of the gene of two rape strains with similar growth periods after inoculation is analyzed through RNAseq, the expression level of the gene at the late flowering stage is determined, and the direct correlation between the expression level of the gene and the sclerotinia rot resistance is found. Two DH lines (R line and S line) with obvious resistance difference are selected, the lesion spots of the two DH lines respectively reach 6.8 cm and 12.1cm, the two copies of BnUGT74B1 gene show difference, and the gene expression level of the resistance lines is increased and reaches obvious level after the sclerotinia sclerotiorum is inoculated for 24 hours.
The BnUGT74B1 gene expression level is directly related to the sclerotinia rot disease resistance of rape, and the method can be used for determining the difference of sclerotinia rot resistance of rape, thereby providing an indication for identifying the genetic difference of sclerotinia rot resistance of rape, improving the accuracy of identifying the resistance of different rape, reducing the error and workload of phenotype identification, and providing a new method for breeding sclerotinia rot resistance of rape.
Drawings
FIG. 1 shows lesion length 7 days after inoculation of two DH lines of stalks;
FIG. 2 is the results of expression level analysis of two copies of BnUGT74B 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.
The normal temperature in the following embodiments of the present invention refers to a natural room temperature condition in four seasons, and is not subjected to additional cooling or heating treatment, and is generally controlled at 10 to 30 ℃, preferably 15 to 25 ℃.
The invention discloses a method for identifying an sclerotinia sclerotiorum resistant rape strain and application thereof. The coding sequence of the BnUGT74B1 gene is shown in SEQ ID NO. 1.
Examples
1. Anti-sense material determination
Two DH lines with similar growth phases were selected for transcriptome sequencing. The scab difference of the two materials is very obvious 7 days after the stalk is inoculated with the sclerotinia sclerotiorum, the scab length is respectively 6.8 cm and 12.1cm, and the scab length is respectively named as R line and S line (figure 1).
2. Transcriptome sequencing
Extracting leaf RNA: the kit was purchased from TRIpure (Cat: RN0102) of Beijing Eldelay Biotechnology Ltd. The specific operation steps are as follows:
1) taking 50-100mg of plant tissue sample, putting the plant tissue sample into a 1.5mL centrifuge tube, and preserving the plant tissue sample in liquid nitrogen;
2) put the sample into mortar, add proper amount of liquid nitrogen, grind rapidly to powder. Transferring the powder into a centrifuge tube with 1.5mL of RNase free;
3) rapidly adding 1mL of TRIpure, mixing uniformly and incubating for 5min at 15-30 ℃;
4) adding 0.2mL of chloroform, tightly covering a centrifugal tube cover, forcibly shaking for 15s, and incubating for 2-3min at 30 ℃;
5) centrifuging at a rotation speed of no more than 12000rpm for 10min at 4 ℃;
6) taking about 0.5-0.6mL of the supernatant into a clean 1.5mL RNase free centrifuge tube, adding 0.5mL of isopropanol, and incubating at 15-30 ℃ for 10 min;
7) centrifuging at 4 deg.C at a speed of 12000rpm for 10min, and removing the upper suspension;
8) add 1mL of 75% ethanol and vortex for several seconds. Centrifuging at a rotation speed of no more than 7500rpm at 4 deg.C for 5 min;
9) the supernatant is decanted, dried for 5-10min, and 30-50. mu.L of DEPC is added to dissolve the RNA.
After RNA extraction, RNA integrity was checked by 1.0% agarose gel electrophoresis and purity and concentration were measured using an Agilent2100 bioanalyzer and a NanoDrop spectrophotometer. Each sampleDiluting 2 μ g total RNA to 50 μ g, and using
TruSeq TM RNA Sample Preparation Kit on RNA samples for library construction, then sent to Huada gene using Illumina-XTen sequencer for all libraries for 2 x 151bp double end sequencing.
3. Analysis of Gene expression levels
And respectively comparing clean reads subjected to NGS QC tool kit quality control to a reference genome of the Brassica napus by using TopHat v2.0.11, and setting parameters by default. Extracting reads which can only be compared to a unique position to calculate the expression quantity of the gene. The expression quantity calculation of the gene and the identification of the differential expression gene are carried out by adopting software Cufflinks v2.2.0. The expression abundance of each gene was expressed as FPKM. Differentially Expressed Genes (DEGs) between inoculated and control samples were screened by analysis of FPKM values.
The results show that the two copies of the BnUGT74B1 gene show difference, and the difference of the expression level of the two copies of the control R mock and the two copies of the control S mock is not significant before the inoculation; the expression levels of both gene copies of the R line were elevated (fig. 2) and reached significant levels compared to the S line 24 hours after inoculation with sclerotinia sclerotiorum; compared with the respective mock control, the two copies of the R line are changed more severely than the S line, and the result indicates that the expression level of the gene is directly related to the resistance to the sclerotinia sclerotiorum, and the expression level of the gene BnUGT74B1 is improved to obviously enhance the resistance to the pathogenic bacteria.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> university of agriculture in Huazhong
<120> method for identifying sclerotinia rot resistant rape strain and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1401
<212> DNA
<213> BnUGT74B1
<400> 1
atggcggaaa caacaacaac aacaacagcg accaactcca aaggccacgt cgtggtctta 60
ccttacccag tccaaggcca cctcaaccca atggtccagt tcgctaaacg cctagtctcc 120
aaaggcgtca aagtcacaat cgccaccacc acctacaccg cctcctccat ctccactccc 180
tccgtctccg tcgaaccaat ctccgacggc cacgacttca tccccatagg cgtccccggc 240
gtcagcatcg acgcatactc cgaatccttc aagctcaacg gctccgaaac cttaacccga 300
gtaatctcaa aattcaaatc cacagattca cccatcgatt cattagtcta cgactctttc 360
ctcccgtggg gactcgaagt cgcgagatct aactccatct cagctgctgc tttcttcacc 420
aacaacctca ccgtttgctc tgttctacgc aaattcgtct ccggtgagtt tcctctcccc 480
gctgatcccg cttccgcgcc gtatctcgtc cgtggcttac cggctttgag ctacgacgag 540
cttccttcct tcgtcggacg tcactcgtcg agccacgcgg agcacgggag agttcttctg 600
aaccagttcc gtaaccacga agatgctgat tggctgttcg tcaacggctt cgaagggtta 660
gagacacaag gttgtgaagt tggagaatca gaggcgatga aggcgacgtt gatcggacct 720
atgataccat ctgcttatct cgacggccga atcaaagacg ataaaggcta cggctcgagc 780
ctgatgaagc cgctctcgga ggagtgtatg gagtggttag acactaagct gagcaagtcg 840
gtggtttttg tttcgtttgg ttcctttggg atcctctttg agaagcaact cgctgaggtg 900
gcaaaggcgt tacaagaatc caactttaac ttcttgtggg tgatcaaaga agctcatata 960
gcgaagttac cagaagggtt tgtggaagct accaaagaca gagcgttgct tgtttcttgg 1020
tgtaaccagc ttgaggtttt agctcatgaa tcgataggtt gctttttgac tcactgcggt 1080
tggaactcga cgttggaagg attgagtttg ggagttccga tggttggtgt gcctcagtgg 1140
agtgatcaga tgaatgatgc taagtttgtg gaggaggttt ggagagttgg gtatagagcg 1200
aaggaggaag ctgggggagg agttgtgaag agcgatgagg tggtgaggtg tttgagagga 1260
gtgatggaag gagagagtag tgtggagatt agagagagtt ctaagaagtg gaaagatttg 1320
gctgtgaagg cgatgagtga aggaggaagc tctgatcgga gcattaatga gtttgtggag 1380
agtctaggga agaaacattg a 1401
Claims (2)
1. A method for identifying an antibacterial Sclerotinia sclerotiorum strain is characterized by comprising the step of detecting the expression level of BnUGT74B1 gene after Sclerotinia sclerotiorum is inoculated at the later flowering stage of rape, wherein the resistance line with the expression level of BnUGT74B1 gene obviously improved is provided.
2. Use of the method of claim 1 for the identification of an sclerotinia sclerotiorum resistant oilseed rape line in the breeding of elite varieties of oilseed rape.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102140446A (en) * | 2011-01-20 | 2011-08-03 | 湖南农业大学 | Application of rape iMyAP gene over-expression in sclerotinia sclerotiorum resistance of rape |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140446A (en) * | 2011-01-20 | 2011-08-03 | 湖南农业大学 | Application of rape iMyAP gene over-expression in sclerotinia sclerotiorum resistance of rape |
Non-Patent Citations (2)
| Title |
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| 田志涛等: "甘蓝型油菜种子和角果皮中硫苷含量的动态变化及转录组关联分析", 《中国农业科学》 * |
| 陈颖: "油菜芳香族芥子油苷抗菌核病作用研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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