Construction and Utilization of Yeast Two-hybrid cDNA Library of Wheat Interacted by Puccinia triticina

doi:10.13560/j.cnki.biotech.bull.1985.2023-0251

Abstract

Abstract:

In order to screen the target proteins interacting with effector proteins of the wheat leaf rust and to lay a foundation for dissecting the defense response mechanism of the effector proteins interfering with hosts and disease resistance breeding, the samples of interaction between the isolate13-5-28-1（JHKT）and susceptible wheat variety Thatcher at 0-12 d were collected. The three-frame homogenization was constructed by SMART method to screen the wheat cDNA library interacting with the wheat leaf rust. The homologous recombination method was used to construct the recombinant bait vector pGBKT7-Pt34084 of the effector factor Pt34084, and it was used as the bait protein to screen the interaction target protein by co-transformation method. The results showed that the cDNA library structure of the interaction between the wheat leaf rust and wheat was successfully constructed. The library capacity was about 1.05×10⁷ CFU/mL, the titer was 1.2×10⁹ CFU/mL, the average insertion fragment was more than 1 kb, and the recombination positive rate was 100%. The cDNA library met the requirement. The constructed bait recombinant vector pGBKT7-Pt34084 inhibited its self-activation after the addition of 20 mmol/L 3-AT and 400 ng/mL ABA to SD/-Trp-His-Ade medium. By this library a total of 16 different candidate interacting proteins from the wheat and the leaf rust were screened. These proteins were involved in plant metabolism, hormone signal transduction, plant disease resistance and other aspects. It indicated that Pt34084 could interfere with the host defense response and promote leaf rust infection through multiple pathways. The results are conducive to analyzing the pathogenic mechanism of wheat leaf rust and lay a foundation for creating new effective strategies to prevent and control wheat leaf rust.

Key words: Puccinia triticina Eriks., wheat leaf rust, cDNA library, yeast two-hybrid, effector protein, interaction protein, screening

WEN Xiao-lei, LI Jian-yuan, LI Na, ZHANG Na, YANG Wen-xiang. Construction and Utilization of Yeast Two-hybrid cDNA Library of Wheat Interacted by Puccinia triticina[J]. Biotechnology Bulletin, 2023, 39(9): 136-146.

Figures/Tables 11

References 47

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编号No.	基因登录号 GenBank No.	候选蛋白 Candidate proteins	来源 Source	一致性 Identity/%
1	XM_044577317.1	光照后叶绿素荧光增加蛋白，类叶绿体 Post-illumination chlorophyll fluorescence increasing protein, chloroplast	小麦 T. aestivum	100.00
2	XM_037619534.1	光系统I组装蛋白2，类叶绿体 Protein photosystem I assembly 2, chloroplast	野生型二粒小麦 T. dicoccoides	99.61
3	XM_044569896.1	过氧化物酶11 Peroxidase 11-like	小麦 T. aestivum	89.11
4	XM_044551498.1	细胞壁连接的类受体激酶 Wall-associated receptor kinase 2-like	小麦 T. aestivum	88.27
5	XM_044521323.1	蛋白酶体组装伴侣4 Proteasome assembly chaperone 4-like	小麦 T. aestivum	99.02
6	XM_044485885.1	富含丝氨酸的无特征蛋白 Uncharacterized serine-rich protein	小麦 T. aestivum	99.35
7	XM_044514591.1	无特征蛋白 Uncharacterized protein LOC123092769	小麦 T. aestivum	99.47
8	XM_037564333.1	无特征蛋白 Uncharacterized protein LOC119285117	野生型二粒小麦 T. dicoccoides	98.90
9	XM_020296657.3	蛋白磷酸酶2C 26 Protein phosphatase 2C 26	山羊草 Aegilops tauschii	99.40
10	XM_003333436.2	假定蛋白 Hypothetical protein	小麦秆锈菌 P. graminis f. sp. tritici	85.12
11	XM_037577936.1	60S 核糖体蛋白 L15-2 60S ribosomal protein L15-2	野生型二粒小麦 T. dicoccoides	99.72
12	XM_020343972.3	无特征蛋白 Uncharacterized protein LOC109785374	山羊草 A. tauschii	99.13
13	XM_020323127.3	E3泛素蛋白连接酶 XBOS34 Probable E3 ubiquitin-protein ligase XBOS34	山羊草 A. tauschii	99.75
14	XM_044474326.1	无特征蛋白 Uncharacterized protein LOC123051461	小麦 T. aestivum	93.80
15	XM_003323195.2	甲硫氨酰氨基肽酶 Methionyl aminopeptidase	小麦秆锈菌 P. graminis f. sp. tritici	88.90
16	XM_044559490.1	含卤酸脱卤酶类水解酶结构域的蛋白质 Haloacid dehalogenase-like hydrolase domain-containing protein At2g33255	小麦 T. aestivum	98.92

编号No.	基因登录号 GenBank No.	候选蛋白 Candidate proteins	来源 Source	一致性 Identity/%
1	XM_044577317.1	光照后叶绿素荧光增加蛋白，类叶绿体 Post-illumination chlorophyll fluorescence increasing protein, chloroplast	小麦 T. aestivum	100.00
2	XM_037619534.1	光系统I组装蛋白2，类叶绿体 Protein photosystem I assembly 2, chloroplast	野生型二粒小麦 T. dicoccoides	99.61
3	XM_044569896.1	过氧化物酶11 Peroxidase 11-like	小麦 T. aestivum	89.11
4	XM_044551498.1	细胞壁连接的类受体激酶 Wall-associated receptor kinase 2-like	小麦 T. aestivum	88.27
5	XM_044521323.1	蛋白酶体组装伴侣4 Proteasome assembly chaperone 4-like	小麦 T. aestivum	99.02
6	XM_044485885.1	富含丝氨酸的无特征蛋白 Uncharacterized serine-rich protein	小麦 T. aestivum	99.35
7	XM_044514591.1	无特征蛋白 Uncharacterized protein LOC123092769	小麦 T. aestivum	99.47
8	XM_037564333.1	无特征蛋白 Uncharacterized protein LOC119285117	野生型二粒小麦 T. dicoccoides	98.90
9	XM_020296657.3	蛋白磷酸酶2C 26 Protein phosphatase 2C 26	山羊草 Aegilops tauschii	99.40
10	XM_003333436.2	假定蛋白 Hypothetical protein	小麦秆锈菌 P. graminis f. sp. tritici	85.12
11	XM_037577936.1	60S 核糖体蛋白 L15-2 60S ribosomal protein L15-2	野生型二粒小麦 T. dicoccoides	99.72
12	XM_020343972.3	无特征蛋白 Uncharacterized protein LOC109785374	山羊草 A. tauschii	99.13
13	XM_020323127.3	E3泛素蛋白连接酶 XBOS34 Probable E3 ubiquitin-protein ligase XBOS34	山羊草 A. tauschii	99.75
14	XM_044474326.1	无特征蛋白 Uncharacterized protein LOC123051461	小麦 T. aestivum	93.80
15	XM_003323195.2	甲硫氨酰氨基肽酶 Methionyl aminopeptidase	小麦秆锈菌 P. graminis f. sp. tritici	88.90
16	XM_044559490.1	含卤酸脱卤酶类水解酶结构域的蛋白质 Haloacid dehalogenase-like hydrolase domain-containing protein At2g33255	小麦 T. aestivum	98.92