Cloning of γ‑Gliadin 1 Gene and Screening of Its Interacting Proteins in Wheat

doi:10.15933/j.cnki.1004-3268.2024.11.003

Abstract

Abstract: In order to further study the molecular mechanism of γ‑gliadin 1 gene in regulating flour quality of wheat,the γ‑gliadin 1 gene was cloned using Zhengmai 158(low protein content and high dough strength)as materials,bait vector pGBKT7‑γ‑gliadin 1 was constructed,the interacting proteins of wheat γ‑gliadin 1 were screened from Zhengmai 158 cDNA library by the yeast two‑hybrid system,and rotary test was used to validate the interaction between key candidate proteins and γ‑gliadin 1.The results showed that the γ‑gliadin 1 gene was successfully cloned with the cDNA length of 1 020 bp.A total of 117 blue clones were screened by the yeast two‑hybrid system.Ten possible candidate interacting proteins of γ‑gliadin 1 were obtained through colony PCR detection,sequencing,and homology analysis using the BLAST on NCBI,which were cysteine‑rich and transmembrane domain‑containing protein 1‑like(XM_044546254.1),alpha‑amylase/trypsin inhibitor‑like(XM_044509330.1),lichenase 2(XM_044550285.1,XM_044594213.1),sucrose 1‑fructosyltransferase‑like(XM_044554537.1),pullulanase 1(XM_044577151.1),ervatamin‑B‑like(XM_044577994.1),cell number regulator CNR8‑like(XM_044483337.1,XM_044491902.1)and CNR2‑like(XM_044468782.1)related to plant reproductive growth(especially development of fruit or grain),and ubiquitin domain‑containing protein DSK2b‑like（dominant suppressor of KAR 2b like,XM_044546840.1)and CIP73(CCa MK‑interacting protein of 73 ku,XM_044554473.1),respectively.The rotation validation assays of candidate interacting proteins indicated that four candidate proteins were probably interacted with γ‑gliadin 1,which were cysteine‑rich and transmembrane domain‑containing protein 1‑like,alpha‑amylase/trypsin inhibitor‑like,lichenase 2 and ervatamin‑B‑like,respectively.Therefore,it is speculated that γ‑gliadin 1 may mainly interact with these candidate proteins to be involved in the synthesis and degradation of storage proteins(such as cysteine)and starch in wheat grains.

Key words: Wheat, γ?gliadin 1, Yeast two?hybrid system, Interacting protein, Rotation validation

摘要： 为进一步研究γ-醇溶蛋白1基因调控小麦面粉品质的分子机制，以郑麦158（低蛋白质含量、高面团强度）为研究材料，克隆小麦γ-醇溶蛋白1基因，构建诱饵载体pGBKT7-γ-醇溶蛋白1，利用酵母双杂交技术从郑麦158的cDNA文库中筛选γ-醇溶蛋白1的候选互作蛋白，并通过回转验证试验进一步验证其互作关系。结果表明，成功克隆了γ-醇溶蛋白1基因，其cDNA全长为1 020 bp。利用酵母双杂交技术共筛选到117个蓝色单克隆菌落，经菌液PCR 检测、测序以及NCBI的BLAST 比对分析，共获得10个与γ-醇溶蛋白1互作的候选蛋白，它们分别是富含半胱氨酸和跨膜结构域蛋白1（XM_044546254.1）、α-淀粉酶抑制剂（XM_044509330.1）、特异性内切-1，3：1，4-β-D-葡聚糖酶2（XM_044550285.1、XM_044594213.1）、蔗糖果糖基转移酶（XM_044554537.1）、支链淀粉酶1（XM_044577151.1）和半胱氨酸蛋白酶B-like（XM_044577994.1），还有与植物生殖生长尤其是果实或者籽粒生长有关的细胞数目调控因子CNR8-like（XM_044483337.1、XM_044491902.1）、CNR2-like（XM_044468782.1）以及参与蛋白质修饰的泛素结构域蛋白DSK2b-like（Dominant suppressor of KAR 2b like，XM_044546840.1）和CIP73（CCa MK‑interacting protein of 73 ku，XM_044554473.1）。候选互作蛋白的回转验证试验结果表明，富含半胱氨酸和跨膜结构域蛋白1、α-淀粉酶抑制剂、特异性内切-1，3：1，4-β-D-葡聚糖酶2和半胱氨酸蛋白酶B-like等4个蛋白质可能与γ-醇溶蛋白1存在互作关系。推测γ-醇溶蛋白1可能与这些蛋白质相互作用参与小麦种子中贮藏蛋白（如半胱氨酸）和淀粉的合成与降解等过程。

关键词: 小麦, γ-醇溶蛋白1, 酵母双杂交, 互作蛋白, 回转验证

CLC Number:

S512

WANG Shasha, HE Ning, HUANG Chao, WANG Gang, WANG Qingchang, CHAO Yueen. Cloning of γ‑Gliadin 1 Gene and Screening of Its Interacting Proteins in Wheat[J]. Journal of Henan Agricultural Sciences, 2024, 53(11): 27-33.

王沙沙, 何宁, 黄超, 王刚, 汪庆昌, 晁岳恩. 小麦γ-醇溶蛋白1 基因克隆及其互作蛋白筛选[J]. 河南农业科学, 2024, 53(11): 27-33.

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