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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (2): 392-401.doi: 10.3724/SP.J.1006.2023.21003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and functional analysis of TaRPP13-1B gene related to powdery mildew resistance in wheat cultivar Brock

LIU Xiao-Ying1(), ZHANG Chi1, WANG Xue-Qing1, YANG Chen-Xiao1, WANG Guang-Yu1, BIAN Yun-Di1, FANG Fang2, WANG Ying2, WANG Zhen-Ying1,*()   

  1. 1College of Life Sciences, Tianjin Normal University, Tianjin Key Laboratory of Animal and Plant Resistance, Tianjin 300387, China
    2Forestry Development Service Center of Baodi District, Tianjin 301899, China
  • Received:2022-01-16 Accepted:2022-05-05 Online:2022-05-25 Published:2022-05-25
  • Contact: WANG Zhen-Ying E-mail:skylxy@tjnu.edu.cn;skywangzy@tjnu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31071671);Tianjin Science and Technology Program(18YFZCNC01100);Tianjin Science and Technology Program(17JCZDJC34100)

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Abstract:

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a severe wheat disease in China. Cloning and pyramiding of different resistance genes to improve crop disease resistance is one of the most cost-effective methods. In this study, TaRPP13-1B on chromosome 1B, which encodes the CC, NB-ARC, and LRR domains, was isolated from common wheat. The relative expression level of TaRPP13-1B in Brock and BJ-1 fluctuated after Bgt inoculation, but the average expression levels were always higher than the susceptible wheat Jing 411. The function of TaRPP13-1B was elucidated by virus-induced gene silencing (VIGS) and overexpression transgenic technique. Silencing of TaRPP13-1B resulted in decreased disease resistance in Brock. Overexpression of TaRPP13-1B improved disease resistance in the transgenic wheat seedlings of Jinqiang 5 cultivar. The above results demonstrated that TaRPP13-1B was involved in the defense response of wheat to powdery mildew, which provided valuable genetic resources for breeding of resistant varieties.

Key words: wheat, NBS-LRR gene, TaRPP13-1B, powdery mildew resistance

Table 1

Primers used in this study"

引物Primer name 序列Sequence (5'-3') 用途Gene usage
TaRPP13-1B-F/R F: ATGGAGTTAGCCGTGGGCGC
R: TCAATCATGATACTCGTTAATAATTAGGG		 cDNA克隆 Isolation of cDNA
RPP13-1B-GSP1 F: GCCTGGGCCAACTACGTTTCAAGTC 3'RACE第1轮PCR 1st run PCR of 3'RACE
RPP13-1B-GSP2 F: AAGCCTTCGAGGGACAGACG 3'RACE 第2轮PCR 2nd run PCR of 3'RACE
RPP13-1B-5RACE-1 R: AATCTCTGTCTCCCTTATGTCTAGGGTC 5'RACE第1轮PCR 1st run PCR of 5'RACE
RPP13-1B-5RACE-2 R: ATGACTTGAAACGTAGTTGGCCCAGG 5'RACE第2轮PCR 2nd run PCR of 5'RACE
TaRPP13-1B-ORF F: ATGGAGTTAGCCGTGGGCGC 全长cDNA克隆 Isolation of full-length cDNA
R: TCAATCATGATACTCGTTAATAATTAGGG
Actin-F/R F: TACTCCCTCACAACAACCG qRT-PCR
R: AGAACCTCCACTGAGAACAA
qRPP13-1B F: CCTACGACATCGAGGACTGC qRT-PCR
R: CGTACAGAGTCCTGCGGATC
RPP13-1B-V F: CTAGCTAGCAAAATGGATTACGGAAGGG BSMVγ: TaRPP13-1B载体构建
Construction of BSMVγ: TaRPP13-1B vector
R: CTAGCTAGCCGACCAGTAGCCACCAAC
RPP13-1B-BglF/BstE R F: GAAGATCTATGGAGTTAGCCGTGGGCGC TaRPP13-1B过表达载体构建
Construction of TaRPP13-1B overexpression vector
R: GGTAACCTCAATCATGATACTCGTTAATAATTAGGG
RPP13-1B-R(35S) TCTTCTGAAAAGTGCTGCCTG 鉴定转基因小麦
Identification of transgenic wheats

Fig. 1

Structure characterization of TaRPP13-1B gene A: predictive schematic for TaRPP13-1B sequence; B: predictive domain schematic for coding TaRPP13-1B protein; C: the phylogenetic tree of diverse RPP13-like proteins family."

Fig. 2

Homologous comparisons of TaRPP13-1B and TaRPP13-3"

Fig. 3

Relative expression level of TaRPP13-1B genes in Bgt-inoculated seedlings Jing 411: wheat susceptible variety; BJ-1: near isogenic line; Brock: wheat resistant variety; Student’s t-test, *: P < 0.05; **: P < 0.01."

Fig. 4

Silencing of TaRPP13-1B reduce wheat resistance against Bgt A: phenotype of Brock leaves inoculated with BSMV; B: TaRPP13-1B-knockdown plants inoculated with BgtE09; C: the relative expression of TaRPP13-1B (*: P < 0.05)."

Fig. 5

Microscopic observation of the spores on the Brock leaves LA: lobed appressoria; SA: slender appressoria; AGT: appressorium germ tube; SH: the secondary hypha; BC: beaded conidia"

Fig. 6

PCR detection of TaRPP13-1B T0 transgenic wheat M: DL2000 marker; 1: control; 2: positive control; 3-14: T0 transgenic wheat."

Fig. 7

Relative expression analysis of TaRPP13-1B in T1 transgenic wheat C: control; 1-11: T1-1-T1-11: transgenic wheat (**: P < 0.01)."

Fig. 8

Overexpression of TaRPP13-1B increase wheat resistance against BgtE09 A: phenotype of transgenic wheat leaves inoculated with BgtE09 for 7 days; B: the lesion area in transgenic wheat leaves (**: P < 0.01)."

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