大豆细胞色素P450家族GmCYP78A71固氮功能解析

doi:10.13304/j.nykjdb.2021.0843

摘要/Abstract

摘要：

大豆可与根瘤菌互作进行共生固氮，有效缓解其对土壤氮素的依赖。细胞色素P450（cytochrome P450，CYP450）是一种血红素单加氧酶，参与植物信号分子、植物激素和黄酮类等多种次生代谢物的合成，为探究其在大豆结瘤固氮中的生物学功能，分析了该家族GmCYP78A71基因的结构，并对其表达模式及生物学功能进行了深入解析。qRT-PCR和转启动子大豆毛根的根瘤GUS染色表明，GmCYP78A71在根瘤中特异表达，且随着根瘤的发育表达量逐渐增高，在成熟根瘤中达到峰值；与对照相比，超表达复合植株的根瘤个数、鲜重、固氮酶活性等显著提高，而RNAi毛状根的根瘤个数、鲜重、固氮酶活性等显著降低。上述结果表明GmCYP78A71对根瘤的生长发育和固氮发挥重要作用。

关键词: 大豆, 共生固氮, 根瘤, 细胞色素单加氧酶, 优异种质

Abstract:

Soybean and rhizobia establish symbiosis in root nodules for symbiotic nitrogen fixation， which effectively relieves its dependence on nitrogen fertilizer in soil. Cytochrome P450 （CYP450）， a heme monooxygenase， is involved in the synthesis and metabolism of many secondary metabolites such as plant signal molecules， plant hormones and flavonoids. In order to explore the function of GmCYP78A71 in nodule formation and nitrogen fixation in soybean， this study analyzed thestructureof GmCYP78A71， and then analyzed its expression pattern and biological function. The results of qRT-PCR and GmCYP78A71 promoter fused GUS staining showed that GmCYP78A71 was specifically expressed in the nodules and the expression abundance increased gradually during nodule development with a peak in mature nodules； the number， fresh weight and nitrogenase activity of nodules of overexpressed transgenic composite plants significantly increased compared with the control， while the number， fresh weight and nitrogenase activity of nodules of RNAi hairy roots significantly decreased. Above results indicated that GmCYP78A71 played an important role in nodule growth and development and nitrogen fixation.

Key words: soybean, symbiotic nitrogen fixation, nodule, cytochrome monooxygenase, elite germplasm

中图分类号:

杨占武, 杜汇, 邢馨竹, 李文龙, 孔佑宾, 李喜焕, 张彩英. 大豆细胞色素P450家族GmCYP78A71固氮功能解析[J]. 中国农业科技导报, 2023, 25(1): 50-57.

Zhanwu YANG, Hui DU, Xinzhu XING, Wenlong LI, Youbin KONG, Xihuan LI, Caiying ZHANG. Functional Analysis of Cytochrome P450 Family GmCYP78A71 in Soybean Nodulation[J]. Journal of Agricultural Science and Technology, 2023, 25(1): 50-57.

图/表 5

表1 本研究所用引物

Table 1 Primers sused in this study

引物名称 Primer name	引物序列 Primer sequence （5’-3’）
Promoter-F	CCCCTGCAGGGCAATGAAAATAATGCCATGTC
Promoter-R	CCCGGATCCGATTAGGAGCACGTAAGATGATTC
RT-F	TGGTTGGTGAAATGGTGATG
RT-R	CCTCTACCACCCCTTTGTCC
ORF-F	CCTCTAGAATGATTCCAACACTTGTTTGTATTG
ORF-R	AAGGATCCTCAAGAGCTTATGGTGTTGAACCTG
RNAi-F	GGGGTACCACTAGTATTGGCGCCACCTACGTAAA
RNAi-R Actin11-F Actin11-R	GGGGATCCGAGCTCCCCAACGCCTCCTTTGTTTG ATCTTGACTGAGCGTGGTTATTCC GCTGGTCCTGGCTGTCTCC

图1 GmCYP78A71基因序列及CYP78A家族表达量分析A：GmCYP78A71基因组序列分析；B：GmCYP78A71蛋白结构域分析；C：CYP78A家族表达量分析

Fig. 1 Sequence analysis of GmCYP78A71 and expression of CYP78A familyA： Genome sequence of GmCYP78A71； B：GmCYP78A71 protein domain analysis； C： Expression analysis of CYP78A family

图2 GmCYP78A71相对表达量A：GmCYP78A71在不同器官的表达量；B：GmCYP78A71在根瘤不同发育时期的表达量

Fig. 2 Relative expression level of GmCYP78A71A： Expression levels of GmCYP78A71 in different organs； B： Expression levels of GmCYP78A71 in different developmental stages of nodule

图3 毛根和不同发育时期根瘤的GUS染色

Fig. 3 GUS staining of roots and nodules at different developmental stages

图4 GmCYP78A71转基因复合植株根瘤表型及生物学功能分析A：根瘤表型；B：GmCYP78A71相对表达量；C：固氮酶活性；D：根瘤数；E：根瘤鲜重。OX—过表达株系；WT—野生型；RNAi—RNAi株系

Fig. 4 Phenotype and biological function analysis of nodules of GmCYP78A71 transgenic complex plantA： Phenotype of nodules； B： Relative expression level of GmCYP78A71； C： Nitrogenase activity； D： Nodule number； E： Nodule fresh weight. OX—Overexpression lines； WT—Wild type； RNAi—RNAi lines

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