油莎豆乙酰乳酸合酶基因CeALS的克隆与分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1198

生物技术通报 ›› 2022, Vol. 38 ›› Issue (4): 184-192.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1198

油莎豆乙酰乳酸合酶基因CeALS的克隆与分析

肖艳华¹^,²(), 邹智²(), 赵永国³, 郭安平², 张丽¹()

1.中南民族大学生命科学学院武陵山区特色资源植物种质保护与利用湖北省重点实验室,武汉 430074
2.中国热带农业科学院三亚研究院热带生物技术研究所海南省南繁生物安全与分子育种重点实验室,海口 571101
3.广东石油化工学院生物与食品工程学院,茂名 525000

收稿日期:2021-09-16 出版日期:2022-04-26 发布日期:2022-05-06
通讯作者: 邹智,男,研究员,研究方向：基因组学与分子生物学;E-mail： zouzhi2008@126.com;
张丽,女,副教授,研究方向：植物分子生物学;E-mail： zhangli0624@mail.scuec.edu.cn
作者简介:肖艳华,女,硕士研究生,研究方向：油莎豆分子育种;E-mail： 1740329672@qq.com
基金资助:
海南省自然科学基金(320RC705);海南省自然科学基金(319MS093);国家自然科学基金项目(31971688);2018年度广东省普通高校特色创新项目(2018KTSCX148)

Molecular Cloning and Characterization of an Acetolactate Synthase Gene（CeALS）from Cyperus esculentus L.

XIAO Yan-hua¹^,²(), ZOU Zhi²(), ZHAO Yong-guo³, GUO An-ping², ZHANG Li¹()

1. College of Life Science,South-Central University for Nationalities,Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China,Wuhan 430074
2. Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions,Institute of Tropical Bioscience and Biotechnology,Sanya Research Institute of Chinese Academy of Tropical Agricultural Sciences,Haikou 571101
3. School of Biological and Food Engineering,Guangdong University of Petrochemical Technology,Maoming 525000

Received:2021-09-16 Published:2022-04-26 Online:2022-05-06

摘要/Abstract

摘要：

乙酰乳酸合酶（ALS）在植物的除草剂抗性中发挥重要作用。为揭示油莎豆ALS编码基因的序列特征、进化关系及表达特性,研究基于转录组数据,应用RT-PCR技术对其进行了克隆。序列分析显示,CeALS预测编码645个氨基酸,理论分子量为69.94 kD、等电点为6.10,属于叶绿体定位的亲水性蛋白。保守结构域和同源分析显示蛋白含有TPP_enzyme_N、TPP_enzyme_M和TPP_enzyme_C三个完整的乙酰乳酸合酶功能域,与其他莎草科植物中同源蛋白的一致性都在90%以上。进化分析显示,莎草科与禾本科属禾本目的姊妹科。qRT-PCR分析显示,CeALS主要在叶片和块茎中表达;在叶片发育过程中呈现逐步递增的趋势,其在成熟和衰老叶片中的表达水平显著高于幼嫩叶片。序列比对和SNP分析显示,团队收集的56份油莎豆种质均不存在抗药性变异。这些结果为除草剂抗性分子育种及油莎豆的开发与利用奠定了坚实的基础。

关键词: 油莎豆, 油料作物, 乙酰乳酸合酶, 除草剂抗性

Abstract:

Acetolactate synthase（ALS）plays an important role in the herbicide resistance of plants. To uncover the sequence characteristics,evolutionary relationship,and expression pattern of the ALS gene in tigernut（Cyperus esculentus L.）,in this study,RT-PCR was adopted to isolate the corresponding gene CeALS on the basis of transcriptome data. Sequence analysis revealed that CeALS putatively encoded 646 amino acids with a theoretical molecular weight of 69.94 kD and an isolectric point of 6.10,which was predicted to be a hydrophilic protein with chloroplast localization. The protein was shown to harbor three conserved domains,i.e.,TPP_enzyme_N,TPP_enzyme_M,and TPP_enzyme_C,which were specific to acetolactate synthase. Homologous analysis suggested that CeALS shared an identity of >90% with homologs from other Cyperaceae plants. Phylogenetic analysis supported that Cyperaceae was a sister family to Poaceae within Poales. qRT-PCR analysis revealed that CeALS was mainly expressed in mature leaves and tubers. During leaf development,an increasing trend was observed and the expression level in mature and senescent leaves was significantly higher than that in young leaves. Sequence alignment and SNP analysis supported that no target site resistance mutations to acetolactate-synthase-inhibiting herbicides were found in 56 germplasms collected by our group. Results obtained in this study lay a solid foundation for molecular breeding for herbicide resistance as well as development and utilization of tigernut.

Key words: Cyperus esculentus L., oil crop, acetolactate synthase, herbicide resistance

肖艳华, 邹智, 赵永国, 郭安平, 张丽. 油莎豆乙酰乳酸合酶基因CeALS的克隆与分析[J]. 生物技术通报, 2022, 38(4): 184-192.

XIAO Yan-hua, ZOU Zhi, ZHAO Yong-guo, GUO An-ping, ZHANG Li. Molecular Cloning and Characterization of an Acetolactate Synthase Gene（CeALS）from Cyperus esculentus L.[J]. Biotechnology Bulletin, 2022, 38(4): 184-192.

图/表 7

图1 CeALS的全长cDNA及其推测编码的氨基酸基因编码区用大写的碱基字母标识,5' UTR和3' UTR用小写的碱基字母标识,基因克隆引物CeALSF/R用单下划线标出,同源重组引物CeALSHF/R的基因序列部分用双下划线标出

Fig.1 Full-length cDNA sequence of CeALS and its dedu-ced coding amino acids The gene coding region is identified by uppercase base letters,the 5' UTR and 3' UTR are identified by lowercase bases,the cloning primer pair CeALSF/R are marked with single underlines,and the primer pair CeALSHF/R for homologous recombinant was marked by double underlines

图2 油莎豆CeALS基因的PCR扩增 A：第一轮PCR扩增结果;B：第二轮PCR扩增结果;M：DNA marker III;1：空白对照;2和3：CeALS

Fig. 2 PCR amplification of CeALS in C. esculentus A：Results of the first round of PCR amplification. B：Results of the second round of PCR amplification. M：DNA marker III. 1：Blank control. 2 and 3：CeALS

表1 拟南芥和20种单子叶植物中ALS基因的结构及其编码蛋白的理化特性

Table 1 Gene structure and physicochemical properties of ALS genes in Arabidopsis and 20 monocots

基因 Gene	内含子数 Number of intron	蛋白 Protein/AA	信号肽 Transit peptide/AA	分子量 MW/kD	等电点 pI	总平均疏水指数 GRAVY	脂肪族指数 AI	不稳定系数 II
AtALS	0	670	55	72.59	6.20	-0.130	87.78	42.93
PeALS	0	657	58	71.51	6.78	-0.126	95.16	40.20
PdALS	0	658	68	70.91	6.17	-0.054	95.06	43.27
EgALS	0	658	68	70.89	6.27	-0.063	94.92	41.18
SpALS	0	659	52	71.81	5.97	-0.138	92.67	43.44
AcALS	0	655	59	70.44	6.37	-0.061	93.89	45.85
MaALS1	0	655	58	71.12	6.64	-0.091	93.11	38.24
MaALS2	0	660	54	71.84	6.43	-0.103	93.03	37.67
OsALS1	0	644	43	69.39	6.48	-0.027	92.13	41.61
OsALS2	0	663	79	71.18	6.30	-0.111	87.59	36.30
HvALS	0	646	46	69.35	6.58	-0.004	92.32	40.49
BdALS1	0	646	46	69.45	6.55	-0.015	92.46	43.17
BdALS2	0	646	46	70.12	6.41	-0.066	92.15	37.31
SvALS	0	643	41	69.39	6.45	-0.053	88.96	42.81
SiALS	0	643	41	69.38	6.45	-0.054	88.96	42.84
SbALS	0	641	40	69.05	6.42	0.000	92.14	40.01
ZmALS1	0	638	39	68.93	6.69	-0.060	91.16	40.26
ZmALS2	0	638	39	68.99	6.77	-0.036	91.33	40.15
ClALS1	1	644	79	70.11	6.45	-0.145	89.35	38.09
ClALS2	1	644	79	70.34	6.80	-0.163	88.59	37.94
CbALS	1	647	55	70.46	5.85	-0.166	88.92	46.54
CeALS	1	645	54	69.94	6.10	-0.156	89.36	44.93
SwALS1	1	645	80	70.24	6.24	-0.144	90.59	41.67
SwALS2	1	645	77	70.26	6.45	-0.163	89.98	41.97
SjALS1	1	645	80	70.22	6.24	-0.140	90.74	41.67
SjALS2	1	645	80	70.26	6.45	-0.163	89.98	41.97
SmALS1	1	648	80	70.58	6.22	-0.143	90.62	41.68
SmALS2	1	645	77	70.21	6.45	-0.157	90.28	41.37

图3 CeALS蛋白的多序列比对和序列特征乙酰乳酸合酶结构域在其两端用实心箭头标识;叶绿体信号肽、TPP_enzyme_N、TPP_enzyme_M和TPP_enzyme_C用线框标识;可引起抗性改变的保守残基用空心箭头标识

Fig. 3 Multiple sequence alignment and sequence features of CeALS protein The acetolactate synthase domain is marked by solid arrows at both ends. Chloroplast transit peptide and domains of TPP_enzyme_N,TPP_enzyme_M,and TPP_enzyme_C are identified by wire frames,whereas conserved residues that can cause resistance changes are identified by hollow arrows

表2 SNP分布情况

Table 2 Distribution of SNPs

位点Site	CeALS	SNP	位点Site	CeALS	SNP	位点Site	CeALS	SNP
120	A	C	435	C	G	1431	T	G
147	C	T	519	C	T	1512	T	A
237	G	A	631	A	C	1596	T	C
255	G	A	681	T	C	1612	T	G
405	A	G	685	C	A	1702	A	G
408	T	C	840	C	T	1749	T	C
411	C	G	945	C	T	1752	T	C
414	C	T	1128	C	T	1767	T	C
417	C	T	1146	G	A	1771	G	T
420	C	A	1404	G	A	1851	G	T

图4 ALS蛋白的进化分析

Fig. 4 Phylogenetic analysis of ALS proteins

图5 CeALS在不同组织中的表达模式

Fig. 5 Expression pattern of CeALS in various tissues

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油莎豆乙酰乳酸合酶基因CeALS的克隆与分析

Molecular Cloning and Characterization of an Acetolactate Synthase Gene（CeALS）from Cyperus esculentus L.

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