Functional Analysis of Carotenoid Cleavage Dioxygenases GmCCD8 in Soybean Nodulation

doi:10.13304/j.nykjdb.2020.1033

Abstract

Abstract:

Soybean interacts with rhizobia in the soil to fix the atmospheric nitrogen. The symbiotic nitrogen fixation process is complex and a series of genes are induced during nodule development. The carotenoid cleavage dioxygenases （CCDs） regulates anabolic metabolism of various plant hormones， pigments and aromatic compounds. In order to investigate the function of CCDs in biological nitrogen fixation， this study cloned GmCCD8 from soybean cultivar ZH15 to analyze its expression pattern and biological function. GmCCD8 was localized in cytoplasm. The expression pattern of GmCCD8 in soybean nodules were analyzed by qRT-PCR and histochemical GUS staining in transgenic soybean hairy roots and its expression increased during nodule development picked at 28^th day. By analyzing composite transgenic soybean root nodules， GmCCD8 was found to affecte nodule fresh weight and nitrogenase activity. Ultrastructure of GmCCD8 RNAi nodules showed that the formation of vesicle， bacteroids and poly?β?hydroxybutyrate （PHB） in the root nodule was significantly reduced. The above results indicated that GmCCD8 was involved in soybean nodule development and nitrogen fixation， which provided functional genes for molecular breeding with efficient nitrogen fixation ability in soybean.

Key words: soybean, nodules, symbiotic nitrogen fixation, carotenoid cleavage dioxygenase, GmCCD8

摘要：

大豆可与根瘤菌共生进行生物固氮，固氮机制受许多基因诱导调控，类胡萝卜素裂解双加氧酶（carotenoid cleavage dioxygenases， CCDs）可调控多种植物激素、色素、芳香类化合物的合成与代谢。为探讨其在大豆生物固氮中的功能，从大豆育成品种中黄15中克隆类胡萝卜素裂解双加氧酶GmCCD8基因，分析了该基因的表达模式及生物学功能。亚细胞定位表明GmCCD8在细胞质中表达。qRT-PCR技术及启动子融合GUS表达载体转化大豆毛状根，发现GmCCD8随大豆根瘤生长发育表达量逐渐上升，且在第28天根瘤中表达量最高。通过构建过表达及RNAi转基因复合植株，验证了GmCCD8影响根瘤发育及固氮能力。利用透射电镜观察根瘤内部结构，发现GmCCD8被干扰后根瘤内泡囊、类菌体和聚β?羟基丁酸盐的含量明显减少。上述结果表明，GmCCD8参与大豆根瘤生长发育及生物固氮过程，为大豆高效固氮分子育种提供了功能基因。

关键词: 大豆, 根瘤, 共生固氮, 类胡萝卜素裂解双加氧酶, 大豆GmCCD8

CLC Number:

S565.1

Xinzhu XING, Zhanwu YANG, Youbin KONG, Wenlong LI, Hui DU, Xihuan LI, Caiying ZHANG. Functional Analysis of Carotenoid Cleavage Dioxygenases GmCCD8 in Soybean Nodulation[J]. Journal of Agricultural Science and Technology, 2022, 24(1): 46-53.

邢馨竹, 杨占武, 孔佑宾, 李文龙, 杜汇, 李喜焕, 张彩英. 大豆类胡萝卜素裂解双加氧酶GmCCD8固氮功能解析[J]. 中国农业科技导报, 2022, 24(1): 46-53.

Figures/Tables 6

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence（ 5’- 3’）
GmCCD8-GFP	F： CTTGTCTAGAGTATGCTTGTTCCTGACATG
GmCCD8-GFP	R： CGGGGGATCCACTCTTTTGGAACCCA
GmCCD8-GUS	F： GATCCACATGATTGATGATATTTTAGTC
GmCCD8-GUS	R： AATGGGTTTACTCCACTTTTCTGCAT
GmCCD8	F： TTCTCTAGAGCTTCCCTGACCGACAAC
GmCCD8	R： ACCTTGTTGTTAGGATGGAAGCTTGTA
Actin11	F：ATCTTGACTGAGCGTGGTTATTCC
Actin11	R：GCTGGTCCTGGCTGTCTCC

Fig.1 Relative expression of GmCCD8 in nodules

Fig.2 Subcellular localization of GmCCD8 in Arabidopsis protoplasts

Fig.3 Histochemical GUS staining of nodules with GmCCD8 promoter in different developmental stages

Fig.4 Index and phenotypic of nodules in GmCCD8 transgenic composite plantsA： Phenotypic analysis； B： Expression of GmCCD8； C： Nitrogenase activity； D： Nodule fresh weight

Fig.5 Nodule ultrastructure of GmCCD8 RNAi at 28 dNote：VE—Vesicle； NB—Nodule bacteroid； PHB—Poly-β-hydroxy-butyrate.

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