玉米ZmGS5基因克隆、分子特性分析及对拟南芥的遗传转化

doi:10.16035/j.issn.1001-7283.2021.01.003

摘要/Abstract

摘要：

高产是玉米育种的重要目标,籽粒大小是决定籽粒产量的重要因子。基于水稻中控制籽粒大小的OsGS5基因编码序列,利用同源克隆方法,获得了玉米ZmGS5基因,其cDNA全长为1 695bp,开放阅读框1 491bp,编码496个氨基酸,含有Peptidase-S10结构域、1个信号肽和1个丝氨酸羧肽酶（serine carboxypeptidases,SCP）类蛋白所特有的催化活性中心,这些都与SCP家族结构特点相符,也与OsGS5相关研究结果相同;磷酸化位点分析结果表明,ZmGS5含有丝氨酸、苏氨酸和酪氨酸等蛋白激酶识别位点。qRT-PCR分析显示ZmGS5在雄穗和叶片中的表达量较高,而在胚及胚乳中的表达量相对较低。除此之外,采用农杆菌介导法,建立了拟南芥的遗传转化体系,得到纯合转基因株系,T₃代转基因拟南芥种子千粒重为0.0169g,较野生型千粒重（0.0139g）高。

关键词: ZmGS5, 基因克隆, 丝氨酸羧肽酶, 分子特性分析, 遗传转化

Abstract:

High yield is an important goal of the maize breeding, grain size is a major determinative factor of high yield. In this study, based on the method of homology cloning, the OsGS5 sequence was used as a template to obtain the ZmGS5 gene. The sequencing result revealed that the full length cDNA of ZmGS5 was 1695bp. The Open Reading Frame (ORF) encoding 496 amino acid was 1491bp. The ZmGS5 protein contained conservative structure domain Peptidase-S10, a signal peptide, an active site of the serine carboxypeptidases, which were all conformed to the structure of serine carboxypeptidase family and also conformed to the OsGS5 related research results. Phosphorylation locus analysis indicated that the protein contained recognition loci of Ser, Thr, Tyr kinases. The result of real-time quantitative PCR showed that ZmGS5 had a much higher expression level in tassel and leaves, and the lower expression level in embryo and endosperm. According to agrobacterium-mediated method, we established genetic transformation system in Arabidopsis and obtained homozygous transgenic lines. The 1000-seed weight of T₃ Arabidopsis (0.0169g) was higher than that of wild type (0.0139g).

Key words: ZmGS5, Gene cloning, Serine carboxypeptidases, Molecular characteristics analysis, Genetic transformation

刘晓丽, 韩利涛, 魏楠, 申飞, 蔡一林. 玉米ZmGS5基因克隆、分子特性分析及对拟南芥的遗传转化[J]. 作物杂志, 2021 (1): 16–25

Liu Xiaoli, Han Litao, Wei Nan, Shen Fei, Cai Yilin. Cloning, Molecular Characteristics Analysis and Genetic Transformation of Arabidopsis thaliana of ZmGS5 Gene in Maize[J]. Crops, 2021(1): 16–25

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引物Primer	引物序列Primer sequence	引物Primer	引物序列Primer sequence
ZmGS5-F	CATATGGCGACAATGGCAGGG	ZmGS5-R	TTGTCATCTGTGTGTGGGAAGC
ZmGS5-3′-GSP1	TGCTGTTGACAAGGCAGTCGTAGCCAA	ZmGS5-3′-GSP2	CGTAGCCAACAGACAGGAACATTTCAG
ZmGS5-5′-GSP1	GATGATGTGTTGGTGTAGGAGAAGCCA	ZmGS5-5′-GSP2	ATTGAGCCAGAGTAGGAGAGGCTTGTG
UPM	CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT	NUP	CTAATACGACTCACTATAGGGC
ZmGS5-qF	CACAAGCCTCTCCTACTCTGGCTCAAT	ZmGS5-qR	TTCGTGGCTCCTATACTGCGGAAACCT

蛋白 Protein	名称 Name	起点 Starting point	终点 End point	E值 E-value
OsGS5	信号肽	1	30	N/A
	简单重复组件	2	14	N/A
	简单重复组件	17	28	N/A
	Peptidase-S10结构域	48	475	1.1e-136
ZmGS5	信号肽	1	37	N/A
	简单重复组件	13	25	N/A
	Peptidase-S10结构域	64	489	8.2e-141
	简单重复组件	313	330	N/A

名称 Name	位点 Site	氨基酸识别位点 Amino acid recognition site	值 Value	名称 Name	位点 Site	氨基酸识别位点 Amino acid recognition site	值 Value	预测 Forecast
ZmGS5	73	SPPVSQFAG	0.939	OsGS5	57	SPAVSQFAG	0.622	丝氨酸Senine
	99	EAQTSPAHK	0.654		83	EAQASPAPE	0.844
	163	GVGFSYTNT	0.780		149	GVGFSYTNT	0.502
	205	EFYISGESY	0.524		191	EFYISGESY	0.610
	296	SAVFSQYQE	0.662		283	NIIFSQYNQ	0.602
	344	IRMFSGYDP	0.997		328	IKMFSGYDP	0.995
ZmGS5	167	SYTNTSSDL	0.748	OsGS5	153	SYTNTSSDL	0.748	苏氨酸Threonine
	463	MTMVTIRGA	0.815		449	MTMVTVRGA	0.769
ZmGS5	184	AEDAYSFLV	0.902	OsGS5	170	AEDAYSFLV	0.902	酪氨酸Tyrosine
	203	SHEFYISGE	0.821		189	DNEFYISGE	0.925
	209	SGESYAGHY	0.936		195	SGESYAGHY	0.936
	222	AELVYDRNK	0.865		208	ADLVYERNK	0.842
	248	LTDDYYDSK	0.906		235	LTDDYYDSK	0.906
	249	TDDYYDSKG	0.980		236	TDDYYDSKG	0.980
	269	SDEVYERIK	0.986		256	SDQVYERIK	0.952
	350	YDPCYSSNA	0.970		334	YDPCYSSYA	0.917
	357	NAEKYFNDA	0.948		341	YAEDYFNKH	0.955

多态性位点 Polymorphic locus	43、44、45	68	132	151	241	421	673	690	757	808	879	1231	1304	1357	1394	1420	1456	1457
cDNA突变位点 cDNA mutation	插入或删除 Insert or delete	T→C	C→T	C→T	C→T	G→A	C→T	C→T	A→C	C→T	G→T	G→C	C→T	C→A	A→G	T→C	C→T	A→G
B73		T	C	C	C	G	C	C	A	T	G	G	C	C	A	T	C	A
Mo17		T	C	C	C	G	C	C	A	T	T	G	C	C	A	T	C	A
178		T	C	C	C	G	C	C	A	T	G	G	C	C	A	T	C	A
郑58		T	C	C	C	G	T	C	C	C	G	C	T	A	G	C	T	G
昌7-2	GTC	C	C	T	T	A	T	C	C	C	G	C	T	A	G	C	T	G
黄C	GTC	C	C	T	T	A	T	T	C	C	G	G	C	C	A	T	C	A
I15016	GTC	C	T	T	T	A	T	T	C	C	G	G	C	C	A	T	C	A
I15018		T	C	C	C	G	C	C	A	T	G	G	C	C	A	T	C	A
I15020		T	C	C	C	G	T	C	C	C	G	C	T	A	G	C	T	G
I15030	GTC	C	C	T	T	A	T	T	C	C	G	G	C	C	A	T	C	A
I15031	GTC	C	C	T	T	A	T	T	C	C	G	G	C	C	A	T	C	A
氨基酸突变位点 Amino acid mutation	插入或删除 Insert or delete	F→L	A→V					T→I			S→I				I→V		T→A
B73		F	A					T			S				I		T
Mo17		F	A					T			I				I		T
178		F	A					T			S				I		T
郑58		F	A					T			S				V		A
昌7-2	S	L	A					T			S				V		A
黄C	S	L	A					I			S				I		T
I15016	S	L	V					I			S				I		T
I15018		F	A					T			S				I		T
I15020		F	A					T			S				V		A
I15030	S	L	A					I			S				I		T
I15031	S	L	A					I			S				I		T

指标Index	B73	Mo17	178	郑58 Zheng 58	昌7-2 Chang 7-2	黄C Huang C	I15016	I15018	I15020	I15030	I15031
百粒重100-grain weight (g)	19.14	28.28	18.99	24.56	23.78	24.77	18.04	19.05	18.52	24.98	24.54
百粒体积100-grain volume (mL)	18.50	24.67	17.33	23.83	21.00	22.50	17.05	18.30	17.93	22.53	23.40
十粒长10-grain length (cm)	9.90	9.92	7.65	9.86	9.55	9.98	7.69	7.85	7.64	10.25	10.30
十粒宽10-grain width (cm)	7.03	8.51	7.61	7.85	7.38	7.71	7.45	7.49	7.57	7.50	7.58
十粒厚10-grain thickness (cm)	5.07	6.01	5.96	5.81	5.94	5.63	5.66	5.89	5.92	5.32	5.28