Plant disease-resistant protein BjMYB9 as well as encoding gene and application thereof

A disease-resistant protein and gene-encoding technology, applied in plant genetic improvement, botany equipment and methods, applications, etc.

Inactive Publication Date: 2016-11-23
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no one has reported that MYB proteins can specifically bind

Method used

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  • Plant disease-resistant protein BjMYB9 as well as encoding gene and application thereof
  • Plant disease-resistant protein BjMYB9 as well as encoding gene and application thereof
  • Plant disease-resistant protein BjMYB9 as well as encoding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Molecular cloning of embodiment 1, BjMYB9

[0032] 1. Plants and growth conditions

[0033] Plants Arabidopsis thaliana (L.) (ecotype Col-0), Nicotiana.benthamiana and Brassica juncea were grown at 22°C (light) / 19°C (dark) and 16h light / 8h dark cycle conditions . Arabidopsis was used for stable genetic transformation, Nicotiana.benthamiana was used for transient expression, and Brassica juncea was used for construction of cDNA library and endogenous gene expression analysis experiments.

[0034] 2. Screening of transcription factors interacting with W-box-like-4 elements by yeast one-hybrid assay

[0035] The mustard seedlings were sprayed with 200 μg / ml fungal elicitor (hexa-Nacetyl-chitohexaose) (Raventos et al., 1995) on the leaves, and the leaves were quickly frozen in liquid nitrogen at 12, 24, 48 and 72 hours after treatment. Leaf samples taken at different time periods were mixed and total RNA was extracted to construct a mustard cDNA library. The vector used...

Embodiment 2

[0039] Example 2, real-time fluorescent quantitative PCR analysis of RNA expression of BjMYB9 gene induced by fungal elicitor

[0040] 1. Material handling

[0041] Leaves of mustard (Brassica juncea) seedlings were sprayed with 200 μg / ml fungal elicitor (hexa-Nacetyl-chitohexaose), and samples were taken at 24, 48, 72 h after treatment and 0 h of the control (untreated), quickly frozen and ground in liquid nitrogen, and used The plant total RNA extraction kit from TIANGEN company extracts the total RNA of mustard, and treats it with DNAase without RNAase contamination to remove the contamination of genomic DNA in the RNA. The first strand of cDNA was synthesized with RNA reverse transcription kit from TIANGEN Company. The first strand of reverse transcription product cDNA was used for real-time quantitative PCR analysis.

[0042] 2. Real-time fluorescent quantitative PCR (qRT-PCR)

[0043] Real-time quantitative PCR was carried out using SYBR Green Premix (Tiangen Company,...

Embodiment 3

[0045] Example 3, BjMYB9 resistance to Botrytis cinerea phenotype detection

[0046] 1. Genetic transformation of Arabidopsis Col-0 by BjMYB9

[0047] The BjMYB9 cDNA was amplified using the primer pair BjMYB9-F2 / BjMYB9-R (see Table 1 for the sequence), sequenced correctly and cloned into the BamH I and Sal I sites of the pCAMBIA1307 vector to obtain the plasmid pCAMBIA1307-BjMYB9 driven by the 35S promoter. Arabidopsis was transformed using the flower apex method and mediated by Agrobacterium EHA 105 (Clough and Bent, 1998). Positive transgenic lines were detected by PCR using primers BjMYB9-F2 / BjMYB9-R. Three independent positive transgenic lines of the T2 generation were screened again in MS medium containing 40 μg / ml hygromycin, and then transplanted into the soil. They were inoculated with Botrytis cinerea when they were about 4 weeks old.

[0048] 2. Cultivation of Botrytis cinerea (B.cinerea) and inoculation treatment of Arabidopsis

[0049] Botrytis cinerea (B. cine...

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Abstract

The invention discloses Brassica juncea disease-resistant protein BjMYB9 as well as an encoding gene sequence and application thereof. The amino acid sequence of the disease-resistant protein BjMYB9 is as shown in SEQ ID NO: 1, and the nucleotide sequence of the encoding gene of the disease-resistant protein BjMYB9 is as shown in SEQ ID NO: 2. A R2R3-MYB type transcription factor capable of being specifically bound with a fungus inducing core element W-box-like-4 in a promoter BjC-P of a Brassica juncea disease-resistant gene BjCHI1 is screened with a yeast one-hybrid technology and is named as BjMYB9. A gel retardation experiment and a tobacco transient expression experiment prove that BjMYB9 can be specifically bound with W-box-like-4 and activates the promoter. Expression of the BjMYB9 gene in Brassica juncea RNA is obviously induced by fungi, the gene can significantly enhance the resistance of arabidopsis thaliana to Botrytis cinerea after overexpression in a model plant arabidopsis thaliana, thereby providing an experimental basis and genetic resource for revealing of the plant disease-resistant mechanism and cultivation of disease-resistant varieties.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a plant disease resistance protein BjMYB9 and its coding gene and application. Background technique [0002] Botrytis cinerea, as the most important plant fungal pathogen, can infect more than 200 kinds of plants, including almost all vegetable and fruit crops, causing losses of 10 billion or even 100 billion US dollars in the world every year, and causing huge losses to agricultural production. economic loss (Dean et al., 2012; Lu et al., 2013; Weiberg et al., 2013). [0003] In order to defend against pathogen infection, plants have evolved complex defense mechanisms to deal with pathogen attacks (Jones and Dangl, 2006; Lu et al., 2013). During the defense process, some plant genes are activated or induced, and many transcription factors (Transcription Factors, TFs) play important roles (Liu et al., 2013; Windram et al., 2012). The WRKY and AP2 / ERF gene families are major transcrip...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
Inventor 赵开军高英贾双伟
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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