Functional Analysis of Cytochrome P450 Family GmCYP78A71 in Soybean Nodulation

doi:10.13304/j.nykjdb.2021.0843

Abstract

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

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

CLC Number:

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.

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.

Figures/Tables 5

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

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

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

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

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

References 30

1	ZHAO B T, DAI A H, WEI H C, et al.. Arabidopsis KLU homologue GmCYP78A72 regulates seed size in soybean [J]. Plant Mol. Biol., 2016, 90(1-2): 33-47.
2	杨红旗,郝仰坤.我国大豆产业回顾、现状与发展对策[J].广东农业科学, 2010, 37(1):188-191.
	YANG H Q, HAO Y K. Histroical review current situation and development countermeasure of Chinese soybean [J]. Guangdong Agric. Sci., 2010, 37(1): 188-191.
3	姚玉波,马春梅,张磊,等.施氮水平对大豆吸收利用氮素及产量的影响[J].东北农业大学学报. 2009, 40(4): 6-10.
	YAO Y B, MA C M, ZHANG L, et al.. Effect of nitrogen levels on absorption and utilization and utilization of nitrogen and yield of soybean [J]. J. Northeast Agric. Univ., 2009, 40(4): 6-10.
4	CONLEY D J, PAERL H W, Howarth R W, et al.. Controlling Eutrophication: Nitrogen and Phosphorus [J]. Science, 2009, 323(5917): 1014-1015.
5	TILMAN D, FARGIONE J, WOLFF B, et al.. Forecasting agriculturally driven global environmental change [J]. Science, 2001, 292(5515): 281-284.
6	KIERS E T, HUTTON M G, DENISON R F. Human selection and the relaxation of legume defences against ineffective rhizobia [J]. Proc. Biol. Soc., 2007, 274(1629): 3119-3126.
7	YAN A M, WANG E T, KAN F L, et al.. Sinorhizobium meliloti associated with Medicago sativa and Melilotus spp. in arid saline soils in Xinjiang, China. [J]. Int. J. Syst. Evol. Microbiol., 2000, 50(5): 1887-1891.
8	PROVOROV N A, TIKHONOVICH I A. Genetic resources for improving nitrogen fixation in legume-rhizobia symbiosis [J]. Genet. Resour. Crop Evol., 2003, 50(1): 89-99.
9	慈恩,高明.生物固氮的研究进展[J].中国农学通报, 2004, 20(1): 25-28.
	CI E, GAO M. Research progress on biological nitrogen fixation [J]. Chin. Agric. Sci. Bull., 2004, 20(1): 25-28.
10	关大伟,李力,岳现录,等.我国大豆的生物固氮潜力研究[J].植物营养与肥料学报, 2014, 2(6): 1497-1504.
	GUAN D W, LI L I, YUE X L, et al.. Study on potential of biological nitrogen fixation of soybean in China [J]. J. Plant Nutr. Fert., 2014, 20(6): 1497-1504.
11	NEBERT D W, NELSON D R, ADESNIK M, et al.. The P450 superfamily: updated listing of all genes and recommended nomenclature for the chromosomal loci [J]. DNA, 1989, 8(1): 1-13.
12	NELSON D, WERCK-REICHHART D. A P450-centric view of plant evolution [J]. Plant J., 2011, 66(1): 194-211.
13	RENSING S A, LANG D, ZIMMER A D, et al.. The physcomitrella genome reveals evolutionary insights into the conquest of land by plants [J]. Science, 2008, 319(5859): 64-69.
14	NELSON D R. Plant cytochrome P450s from moss to poplar [J]. Phytochem. Rev., 2006, 5(2-3): 193-204.
15	ANASTASIOU E, KENZ S, GERSTUNG M, et al.. Control of plant organ size by KLUH/CYP78A5-dependent intercellular signaling [J]. Dev. Cell, 2007, 13(6): 843-856.
16	FANG W J, WANG Z B, CUI R F, et al.. Maternal control of seed size by EOD3/CYP78A6 in Arabidopsis thaliana [J]. Plant J., 2012, 70(6). 929-939.
17	MAEDA S, DUBOUZET J G, KONDOU Y, et al.. The rice CYP78A gene BSR2 confers resistance to Rhizoctonia solani and affects seed size and growth in Arabidopsis and rice [J/OL]. Sci. Rep., 2019, 9(1), 587 [2021-08-10]. .
18	ZONDLO S C, IRISH V F. CYP78A5 encodes a cytochrome P450 that marks the shoot apical meristem boundary in Arabidopsis [J]. Plant J., 1999, 19(3): 259-268.
19	TAKASHI N, MISAKI K, HIROAKI U, et al.. Highly pleiotropic functions of CYP78As and AMP1 are regulated in non-cell-autonomous/organ-specific manners [J]. Plant Physiol., 2021, 186(1): 767-781.
20	KATSUMATA T, FUKAZAWA J, MAGOME H, et al.. Involvement of the CYP78A subfamily of cytochrome P450 monooxygenases in protonema growth and gametophore formation in the moss Physcomitrella patens [J]. Biosci. Biotechnol. Biochem., 2011, 75(2): 331-336.
21	DAI A H, YANG S X, ZHOU H K, et al.. Evolution and expression divergence of the CYP78A subfamily genes in soybean [J/OL]. Genes, 2018, 9(12): 611 [2021-08-10]. .
22	GUTTIKONDA S K, TRUPTI J, BISHT N C, et al.. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases [J]. BMC Plant Biol., 2010, 10(1): 243-243.
23	KENNETH J L, THOMAS D S. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method [J]. Methods, 2000, 25(4): 402-408.
24	邢馨竹,杨占武,孔佑宾,等.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.
	XING X Z, YANG Z W, KONG Y B, et al.. Fuctional analysis of carotenoid cleavage dioxygenases GmCCD8 in soybean nodulation [J]. J. Agric. Sci. Technol., 2022,24(1):46-53.
25	ZHAO Y J, CHENG Q Q, SU P, et al.. Research progress relating to the role of cytochrome P450 in the biosynthesis of terpenoids in medicinal plants [J]. Appl. Microbiol. Biotechnol., 2014, 98(6): 2371-2383.
26	HULL A K, VIJ R, CELENZA J L. Arabidopsis cytochrome P450s that catalyze the first step of tryptophan-dependent indole-3-acetic acid biosynthesis [J]. Proc. Natl. Acad. Sci. USA, 2000, 97(5): 2379-2384.
27	QI X L, LIU C L, SONG L L, et al.. PaCYP78A9, a cytochrome P450, regulates fruit size in sweet cherry (Prunus avium L.) [J/OL]. Front. Plant Sci., 2017, 8:2076 [2021-08-10]. .
28	HAYASHI S, REID D E, LORENC M T, et al.. Transient Nod factor-dependent gene expression in the nodulation-competent zone of soybean (Glycine max [L.] Merr.) roots [J]. Plant Biotechnol. J., 2012, 10(8): 995-1010.
29	APPLEBY C A, TURNER G L, Macnicol P K. Involvement of oxyleghaemoglobin and cytochrome P-450 in an efficient oxidative phosphorylation pathway which supports nitrogen fixation in Rhizobium [J]. Biochim. Biophys. Acta, 1975, 387(3): 461-474.
30	JUNG W, YU O, LAU S M C, et al.. Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes [J]. Nat. Biotechnol., 2000, 18(2): 208-212.

[1]	Danrong YU, Ziqi SHANG, Shizuo QIU, Fengjing SONG, Zhihui YU, Xiaoyu ZHANG, Xiaoyan ZHANG. Comparative Analysis of Nutritional Composition and Functional Properties of Different Types of Bean Powder [J]. Journal of Agricultural Science and Technology, 2025, 27(8): 144-154.
[2]	Huanbin ZHENG, Ming LI, Suxin YANG, Weilin WU. Alkaline Tolerance Screening and Evaluation Analysis of Soybean Germplasm Resources at Seedling Stage [J]. Journal of Agricultural Science and Technology, 2025, 27(7): 54-71.
[3]	Lei JI, Xiaodong JIANG, Yuanqin SUN, Tianhong LIU, Hongyan LI, Xiao LI, Ying WANG, Shuaizhong ZHANG. Effects of Soybean Oligopeptide and Transaminase on Quality and Protein Physicochemical Properties of Rainbow Trout （Oncorhynchus mykiss） After Hot Boiling [J]. Journal of Agricultural Science and Technology, 2025, 27(4): 149-156.
[4]	Bing YANG. Research Progress on Pathogenic Mechanism， Host Molecule Response， and Prevention and Control Methods of Soybean Phytophthora sojae [J]. Journal of Agricultural Science and Technology, 2025, 27(3): 133-142.
[5]	Na LI, Hua ZHANG, Xinzhu XING, Zhenqi SHAO, Zhanwu YANG, Xihuan LI, Caiying ZHANG. Function Analysis of Soybean Expansin Gene GmEXLA1 inPlantPod and Seed Development [J]. Journal of Agricultural Science and Technology, 2025, 27(3): 49-59.
[6]	Guoqiang DUN, Xingpeng WU, Xinxin JI, Fuli ZHANG, Wenyi JI, Yongzhen YANG. Simulation and Optimization of Soybean Plot Metering Device with Double Swing Plate [J]. Journal of Agricultural Science and Technology, 2024, 26(6): 82-90.
[7]	Wenyue WANG, Zhipeng YAO, Yang YU, Yiqiang GE. Scientific and Technological Innovation of Soybean Seed Industry in China：Current Situation and Strategy [J]. Journal of Agricultural Science and Technology, 2024, 26(3): 1-6.
[8]	Haosen YANG. Impact of Industrialization of Soybean Bio-breeding in China on Trade Dependency [J]. Journal of Agricultural Science and Technology, 2024, 26(11): 15-22.
[9]	Jianmin YAO, Junkui MA, Zhongxiang WANG, Xinyuan BI, Ruizhen LI, Ruiping YANG, Zhao LIU, Fenghui GUO. Application Effect of Full Biodegradable Water Permeable Plastic Film in Soybean-Maize Belt Composite Planting [J]. Journal of Agricultural Science and Technology, 2023, 25(9): 178-185.
[10]	Rui TIAN, Hua ZHANG, Meihong HUANG, Zhenqi SHAO, Xihuan LI, Caiying ZHANG. Mining of Candidate Genes and Genetic Loci Conferring Drought Tolerance in Soybean [J]. Journal of Agricultural Science and Technology, 2023, 25(9): 69-82.
[11]	Chenyang ZHANG, Minggang XU, Fei WANG, Ran LI, Nan SUN. Effects of Manure Application on Soybean Yield and Soil Nutrients in China [J]. Journal of Agricultural Science and Technology, 2023, 25(8): 148-156.
[12]	Boyang KE, Wenlong LI, Caiying ZHANG. Expressions of SWEET Genes During Pod and Seed Developments and Under Different Stress Conditions in Soybean [J]. Journal of Agricultural Science and Technology, 2023, 25(8): 33-52.
[13]	Yaqian SUN, Shiliang CHEN, Jiahao CHU, Xihuan LI, Caiying ZHANG. Mining of QTLs and Candidate Genes for Pod and Seed Traits via Combining BSA-seq and Linkage Mapping in Soybean [J]. Journal of Agricultural Science and Technology, 2023, 25(7): 29-42.
[14]	Jian HU, Gang CHE, Lin WAN, Huiru ZHOU, Guang LI. Study of Soybean Row Line Extraction Method Under High Light Conditions [J]. Journal of Agricultural Science and Technology, 2023, 25(5): 106-111.
[15]	Zhenqi SHAO, Wenlong LI, Youbin KONG, Hui DU, Zhanjun LI, Xihuan LI, Caiying ZHANG. Identification of Soybean Quality Traits and Screening of Elite Germplasms in Huang-Huai-Hai Ecoregion [J]. Journal of Agricultural Science and Technology, 2023, 25(11): 58-69.