CN110734911A - Application of miR159b in regulation and control of rice bacterial leaf blight resistance - Google Patents
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Abstract
The invention discloses application of miR159b in regulation of bacterial blight resistance of rice, provides RNA (namely rice miR159 b) as shown in sequence 1 of a sequence table, also provides DNA molecules as shown in sequence 3 of the sequence table, and also protects a method for preparing a transgenic plant by .
Description
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to application of miR159b in regulation and control of rice bacterial leaf blight resistance.
Background
The rice bacterial leaf blight is which is most distributed in the world and has the most serious damage to the environment caused by infection of gram-negative Xanthomonas (Xanthomonas Oryzae pv. Oryzae, Xoo), can reduce the yield of the rice by 20-30%, can reach 50% even absolutely in severe cases, and can influence the rice quality and the edibility.
The miRNA is -type evolutionarily conserved non-coding single-stranded small-molecule RNA with the length of 21-24nt, has a biological function in plants, mainly regulates the expression of target genes at the level of posttranscriptional negative control, and can participate in the whole life process of plants.
Disclosure of Invention
The invention aims to provide application of miR159b in regulation and control of rice bacterial leaf blight resistance.
The invention provides RNAs, namely rice miR159b, which is shown as a sequence 1 in a sequence table.
The invention also provides RNAs, namely precursor RNA of rice miR159b, which is shown as a sequence 2 in a sequence table.
The invention also provides DNA molecules, namely a coding region of the rice miR159b gene, which is shown as the 295-th and 482-th nucleotides of a sequence 3 in a sequence table.
The invention also provides DNA molecules which are shown as a sequence 3 in a sequence table.
The invention also protects the application of the RNA of any in cultivating a plant resisting bacterial blight, wherein the plant is a gramineae plant, the plant is a rice plant, the plant is rice, and the plant is japonica rice, such as Taibei rice 309.
The invention also protects the application of the RNA of any in regulating and controlling the bacterial leaf blight disease resistance of plants, wherein the regulation is positive regulation, and the positive regulation means that the RNA level is increased, and the bacterial leaf blight disease resistance is improved, the plants are gramineae, the plants are rice, the plants are japonica rice, and the plants are japonica rice, such as Taibei rice 309.
The invention also protects the application of the DNA molecule of any in cultivating a plant resisting bacterial blight, wherein the plant is a gramineae plant, the plant is a rice plant, the plant is japonica rice, and the plant is japonica rice, such as Taibei 309.
The invention also provides a method for preparing transgenic plant, which comprises the following steps of overexpressing the RNA of any in a plant to obtain the transgenic plant with improved bacterial blight resistance, wherein the plant is gramineae, the plant is oryza, the plant is japonica, such as taipei 309.
The invention also provides an method for cultivating plants resistant to bacterial blight, which comprises the following steps of introducing the DNA molecule of any above into a recipient plant to obtain a transgenic plant with enhanced bacterial blight resistance, introducing the DNA molecule into the recipient plant through a plant expression vector and expressing the DNA molecule, wherein the plant expression vector can be a vector pCAMBIA1300-Ubi, and specifically, introducing the DNA molecule into the recipient plant is realized by introducing a recombinant plasmid into the recipient plant, wherein the recombinant plasmid can be obtained by inserting the DNA molecule shown in sequence 3 of a sequence table into a multiple cloning site (for example, between SpeI and Kpn I enzyme cutting sites) of the vector pCAMBIA1300-Ubi, the recipient plant is a rice plant, the recipient plant is japonica rice, and the recipient plant is rice, such as Nichis japonica rice 309.
The present invention also protects the use of the method of any of above in plant breeding for the purpose of obtaining a plant with increased resistance to bacterial blight, the plant is a gramineae, the plant is a rice, the plant is rice and the plant is japonica rice, such as taibei 309.
The bacterial blight of rice mentioned in any above can be caused by Xanthomonas oryzae.
The bacterial blight of rice described in any of above may specifically be caused by a rice pathogenic variety of Xanthomonas oryzae.
The rice bacterial leaf blight of any above can be specifically caused by Philippine bacterial leaf blight physiological race PXO 86.
The invention finds that the miR159b overexpression in rice can improve the resistance of rice to bacterial blight obviously. The miR159b related to the bacterial leaf blight resistance of rice can be used in the fields of improvement of rice germplasm resources and genetic breeding, can also be used for breeding rice varieties with bacterial leaf blight resistance, and has good market application prospects. The discovery of miRNA supplements the understanding of researchers to the rice gene regulation mechanism, and shows a multilevel and multidirectional regulation system of intracellular gene expression. The regulation and control effect of miRNA on bacterial leaf blight of rice is deeply understood, and clues and ideas are provided for improving rice yield and cultivating new germplasm resources.
Drawings
FIG. 1 is the results of the relative expression levels of the miR159b gene in example 1.
FIG. 2 is the results of the relative expression levels of the miR159b gene in example 2.
FIG. 3 is the result of phenotypic identification in example 2; scale bar 1.5cm, indicates a significant level of t-test difference P-value of up to 0.01 compared to WT expression.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, unless otherwise specified, were set up in triplicate and the results averaged.
The rice Taibei 309 (indicated by TP309 or WT), also called Taipei 309 or wild rice, belongs to japonica rice. The following documents are described: song WY, Wang GL, Chen LL, Kim HS, Pi LY, Holsten T, Gardner J, Wang B, ZHai WX, Zhu LH, Fauquet C, Ronald P. (1995). A receiver kinase-like protein encoded by the device disease resistance gene, Xa21.science270,1804-1806.
The rice IRBB5 belongs to the field of indica rice and rice bacterial leaf blight disease-resistant variety. The following documents are described: jiang GH, XiazH, Zhou YL, Wan J, Li DY, Chen RS, ZHai WX and Zhu LH, testing the bacteria resistance gene Xa5 in its functions and functions relating to Xa5(Xa5) by y composing with it's homolog IIA γ 1.Mol Genomics.2006,275(4): 354-.
The rice IR24 belongs to the susceptible varieties of indica rice and rice bacterial leaf blight. The following documents are described: jiang GH, XiazH, Zhou YL, Wan J, Li DY, Chen RS, ZHai WX and Zhu LH, testing the bacteria resistance gene Xa5 in its functions and functions relating to Xa5(Xa5) by y composing with it's homolog IIA γ 1.Mol Genomics.2006,275(4): 354-.
The bacterial blight of rice used in the examples is Philippine bacterial blight physiological race PXO 86.
The vector pCAMBIA1300-Ubi is described in the literature as "pCAMBIA 1300 drive n bvaizeubiptin promoter". The following documents are described: chen SH, Zhou LJ, Xu P, Xue HW. SPOC domain-relating protein Leaf inclusion 3 interactions with LIP1 to rule relationship with root extraction signalling PLoS Genet.2018,14(11) e1007829.doi:10.1371/journal.
In the examples, the reverse transcription was carried out using a reverse transcription kit of OMEGA, Inc. under the trade name R6842-01.
The rice miR159b is shown in a sequence 1 of a sequence table. The precursor RNA of the rice miR159b is shown as a sequence 2 in a sequence table, and the coding region of the rice miR159b gene is shown as a 295-482 th nucleotide in the sequence 3 in the sequence table.
Example 1 expression characteristics of miR159b under rice bacterial blight stress
The method comprises inoculating rice leaf blight bacteria to tillering plant (rice IR24 or rice IRBB5) by artificial leaf cutting inoculation. Inoculating for 0h, 8h, 12h, 24h and 48h respectively, taking leaves, and extracting total miRNA. And carrying out reverse transcription on the total miRNA to obtain cDNA. And (3) detecting the relative expression level of the miR159b gene (U6 gene is used as an internal reference gene) by using cDNA as a template and using stem-loop real-time qPCR.
The primer pairs for identifying the U6 gene are as follows:
U6-qRT-F：5'-GGGGACATCCGATAAAATTGG-3'；
U6-qRT-R：5'-ACCATTTCTCGATTTGTGCGT-3'。
primer pairs for identifying the miR159b gene are as follows:
miR159b-qRT-F：5'-CGGCGGTTTGGATTGAAGGGA-3'；
miR159b-qRT-R：5'-GTGCAGGGTCCGAGGT-3'。
the relative expression level results of the miR159b gene are shown in fig. 1. In the rice bacterial leaf blight disease-resistant material (rice IRBB5), the expression level of the rice miR159b gene is obviously increased when the rice bacterial leaf blight disease-resistant material is inoculated with the rice bacterial leaf blight disease for 0-24 h. In the rice bacterial leaf blight infection material (rice IR24), the expression level of the rice miR159b gene has no obvious change rule.
Example 2 obtaining and identifying transgenic Rice
construction of recombinant plasmid
1. The genomic DNA of the leaves of the rice TP309 is used as a template, a primer pair consisting of miR159b-FP and miR159b-RP is adopted for PCR amplification, and an amplification product is recovered.
miR159b-FP：5'-GACTAGTACCTTTCACCCATCAACAGCA-3'；
miR159b-RP：5'-CGGGGTACCAGTGGCTGAAGTAACGCCAA-3'。
2. Taking the amplification product obtained in the step 1, carrying out double enzyme digestion by using restriction enzymes SpeI and Kpn I, and recovering the enzyme digestion product.
3. The vector pCAMBIA1300-Ubi is subjected to double enzyme digestion by using restriction enzymes SpeI and Kpn I, and a vector skeleton of about 10kb is recovered.
4. And (3) connecting the enzyme digestion product obtained in the step (2) with the vector skeleton obtained in the step (3) to obtain a recombinant plasmid pUbi: (miR) 159 b. According to the sequencing result, the structure of the recombinant plasmid pUbi: miR159b is described as follows: the DNA molecule shown in sequence 3 of the sequence table is inserted between SpeI and Kpn I restriction sites of the vector pCAMBIA 1300-Ubi.
II, obtaining miR159b overexpression plants
1. The recombinant plasmid pUbi:: miR159b is introduced into agrobacterium tumefaciens EHA105 to obtain the recombinant agrobacterium tumefaciens.
2. Preparing an infection solution by using the recombinant agrobacterium obtained in the step 1, then soaking the embryogenic callus of the Taipei 309 of the paddy rice by using the infection solution, and then sequentially carrying out co-culture, screening culture (carrying out two-time screening, wherein the -time screening adopts 50mg/L hygromycin B and 50mg/L carbenicillin, the second-time screening adopts 400mg/L hygromycin B and 400mg/L carbenicillin), differentiation culture and rooting culture to obtain a T0 generation regenerated plant.
3. Taking leaves of T0 generation regenerated plants, extracting genome DNA, and carrying out PCR identification by adopting a primer pair consisting of hpt-F and hpt-R to obtain a positive plant of about 845bp amplification products, wherein the positive plant is a transgenic plant.
hpt-F：5'-TAGGAGGGCGTGGATATGTC-3'；
hpt-R：5'-TACACAGCCATCGGTCCAGA-3'。
4. T0 transgenic plants are selfed, seeds are harvested and cultivated into plants, namely T1 plants, T1 plants are selfed, seeds are harvested, namely T2 seeds, T2 seeds grow into plants, namely T2 plants, the T1 plants are subjected to PCR identification, the method is the same as step 3, for a certain T0 plant, if the T1 plants obtained by selfing are all determined to be positive by PCR, selfed progeny of the T0 plants are homozygous transgenic lines, and two transgenic lines, namely an miR159b OE # 2 line and an miR159b OE # 4 line, are obtained.
Thirdly, detecting the relative expression level of the miR159b gene
The test plants were: the rice is Taipei 309 plants, T2 generation plants of miR159b OE # 2 strain, and T2 generation plants of miR159b OE # 4 strain.
Taking the leaves of the tested plants and extracting the total miRNA. And carrying out reverse transcription on the total miRNA to obtain cDNA. And (3) detecting the relative expression level of the miR159b gene (U6 gene is used as an internal reference gene) by using cDNA as a template and using stem-loop real-time qPCR.
The primer pairs for identifying the U6 gene are as follows:
U6-qRT-F：5'-GGGGACATCCGATAAAATTGG-3'；
U6-qRT-R：5'-ACCATTTCTCGATTTGTGCGT-3'。
primer pairs for identifying the miR159b gene are as follows:
miR159b-qRT-F：5'-CGGCGGTTTGGATTGAAGGGA-3'；
miR159b-qRT-R：5'-GTGCAGGGTCCGAGGT-3'。
the relative expression level results of the miR159b gene are shown in fig. 2. Compared with wild rice, the expression level of the miR159b gene in the miR159b OE # 2 strain and the miR159b OE # 4 strain is obviously increased.
Fourth, phenotype identification
Test seeds: the hybrid rice is obtained by the following steps of (1) Taipei 309 seeds of rice, T2 generation seeds of miR159b OE # 2 strain, and T2 generation seeds of miR159b OE # 4 strain.
Planting the test seeds in field, normally cultivating and managing to tillering stage, inoculating rice bacterial leaf blight by artificial leaf cutting method, inoculating for 5 days, collecting inoculated leaves, and performing DAB staining (for detecting H)2O2Production and content) of inoculated leaf discs and the phenotype was recorded 14 days after inoculation by measuring lesion length of inoculated leaf discs.
Photographs of the leaves 14 days after inoculation are shown in FIG. 3A. The lesion length results are shown in FIG. 3B (15 plants per line). Compared with wild rice, the lesion of the transgenic rice with miR159b overexpression is obviously shortened.
The DAB staining is shown in FIG. 3C. While there was a small amount of brown precipitate near the wound on the inoculated leaves of wild type rice, there was a large amount of brown precipitate near the wound on inoculated leaves of miR159b overexpressing plants. The result shows that the transgenic plant over-expressed by miR159b generates stronger immune response after the bacterial blight infection.
SEQUENCE LISTING
<110> institute of genetics and developmental biology of Chinese academy of sciences
Application of <120> miR159b in regulation and control of bacterial leaf blight resistance of rice
<130>GNCYX192434
<160>3
<170>PatentIn version 3.5
<210>1
<211>21
<212>RNA
<213>Oryzae sativa
<400>1
uuuggauuga agggagcucu g 21
<210>2
<211>188
<212>RNA
<213>Oryzae sativa
<400>2
gguuaugaag uggagcuccu uucguuccaa ugaaagguuu aucugaaggg ugauacagcu 60
gcuuguucau gguucccacu auucuaucuc auaggaaaag agauaggcuu gugguuugca 120
ugaccaagga gccgaaucaa cuccuugcug accacucuuu ggauugaagg gagcucugca 180
ucuugauc 188
<210>3
<211>695
<212>DNA
<213>Oryzae sativa
<400>3
acctttcacc catcaacagc aacaaacaat agttcttttc tttctctctc tccctttgta 60
ggtgtagatg ttgatttcat ggctcttctg atcttcgtgt tctagtctct cagtattata 120
tgcatagttg aagttttttt tggttaacat agtatttctg aagcctctga tgtttgcttt 180
tgctattgca aatcttctag ttttgtttat gctatatact atatgtttca gatctctctt 240
ggtttgtggt tttgtttctt tgttggttgt ttcttgaata tgtggttgcg ttttggttat 300
gaagtggagc tcctttcgtt ccaatgaaag gtttatctga agggtgatac agctgcttgt 360
tcatggttcc cactattcta tctcatagga aaagagatag gcttgtggtt tgcatgacca 420
aggagccgaa tcaactcctt gctgaccact ctttggattg aagggagctc tgcatcttga 480
tcccatatag tagtacatgt cactactgat tgcatgtctt atatatttgc cccctgttca 540
tgttggattt gattttttcc tggttcccca tgtgtagatc taatgatttt tgataatgct 600
tgtacttcaa accagtcata tatagcacct gcatattcga tggagagatg atctggtttt 660
gcaagcttcg ttgttttggc gttacttcag ccact 695
Claims (10)
1, RNAs are shown as a sequence 1 in a sequence table.
2, RNAs are shown as a sequence 2 in a sequence table.
3, DNA molecules, as shown in the 295-482 nucleotides of the sequence 3 of the sequence table.
4, DNA molecules shown in sequence 3 of the sequence table.
5. Use of the RNA according to claim 1 or the RNA according to claim 2 for breeding plants resistant to bacterial blight.
6. Use of the RNA of claim 1 or the RNA of claim 2 for modulating disease resistance to bacterial blight in plants.
7. Use of the DNA molecule of claim 3 or the DNA molecule of claim 4 for growing plants resistant to bacterial blight.
A method of producing a transgenic plant comprising the step of overexpressing in a plant the RNA of claim 1 or the RNA of claim 2 to obtain a transgenic plant having increased bacterial blight resistance.
A method of breeding a plant resistant to bacterial blight comprising the step of introducing the DNA molecule of claim 3 or the DNA molecule of claim 4 into a recipient plant to obtain a transgenic plant having increased bacterial blight resistance.
10. Use of the method of claim 8 or 9 in plant breeding.
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| CN108795971A (en) * | 2018-05-04 | 2018-11-13 | 华南农业大学 | Applications of the miR159 in changing plant root species form |
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| CN108795971A (en) * | 2018-05-04 | 2018-11-13 | 华南农业大学 | Applications of the miR159 in changing plant root species form |
Non-Patent Citations (4)
| Title |
|---|
| JONES-RHOADES, MW等: "Computational Identification of Plant MicroRNAs and Their Targets, Including a Stress-Induced miRNA", 《MOLECULAR CELL》 * |
| KOZOMARA，A.等: "Oryza sativa Japonica Group microRNA osa-miR159a.1", 《NCBI》 * |
| LI，Y.等: "Oryza sativa precursor microRNA 159b gene, complete sequence", 《NCBI》 * |
| ZHAO, YT等: "Dynamic and Coordinated Expression Changes of Rice Small RNAs in Response to Xanthomonas oryzae pv. oryzae", 《JOURNAL OF GENETICS AND GENOMICS》 * |
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