山药DoIPTs基因克隆及珠芽发芽期表达分析

doi:10.7668/hbnxb.20192488

摘要/Abstract

摘要：

为了克隆山药异戊烯基转移酶基因,预测该基因编码蛋白的结构和特性,并检测该基因在山药珠芽发芽期的表达特征,采用同源克隆的方法克隆山药IPT 基因,使用生物信息学的方法预测蛋白质的序列和结构,通过实时荧光定量PCR检测基因的表达量。结果表明,在山药中克隆到了长为1 398,1 044,1 349 bp的3条核酸序列,依次命名为DoIPT1、DoIPT5a和DoIPT5b,基因登录号依次是:MW353160、MW353016、MW353017,各基因的阅读框长度依次为1 137,861,1 011 bp,编码氨基酸长度分别为378,286,336 aa。分析氨基酸生物信息得知,DoIPTs属于不稳定外在膜亲水性蛋白,DoIPT5b存在2个信号肽,其余无信号肽;3个蛋白的二级结构预测与三级结构模型图相符;DoIPTs的编码区与其他植物的异戊烯基转移酶蛋白高度同源。DoIPTs编码区域显示,DoIPTs N端含有P-loop NTPase结构域GXXGXGKS(T),具有催化形成细胞分裂素的能力。实时荧光定量PCR分析结果表明,在22 ℃无外界水条件下珠芽发芽时表皮中DoIPT5a的表达量最高,在潮湿环境下DoIPT1和DoIPT5b基因的表达量最高;多效唑处理能够使DoIPTs基因的表达上调。结果为进一步研究山药DoIPTs基因的功能奠定了基础。

关键词: 山药, 基因克隆, DoIPT基因, 珠芽, 多效唑, 相对定量表达

Abstract:

The aim of this research was to clone IPT(Isopentenyl transferase,IPT ) genes in yam,predict the structure and property of DoIPT proteins and identify the expression of DoIPTs during bulbil sprouting.DoIPTs were cloned by homologous cloning techniques.The structure and property of DoIPT proteins were predicted by bioinformatics analysis.Real-time PCR technique was applied to identify the expression of these genes.Three nucleic acid sequences of 1 398,1 044,1 349 bp were obtained from yam.The three sequences were named DoIPT1,DoIPT5a and DoIPT5b,and the registration numbers of these genes were MW353160,MW353016 and MW353017,respectively.The reading frame lengths were 1 137,861,1 011 bp respectively,and the encoded amino acid lengths were 378,286,336 aa,respectively.Bioinformatics analysis showed that DoIPTs were unstable,hydrophilic and external family proteins.There were two signal peptides in DoIPT5b,and no signal peptide was found in the other two proteins.The secondary structures in the three proteins were consistent with the prediction models of the tertiary structures of three genes.The encoded regions of DoIPTs had high similarity with the isopentenyl transferase protein of other plants.Coding areas of DoIPTs showed that the N-terminal regions of DoIPTs contained the P-loop NTPase structural domain GXXGXGKS(T),which was able to catalyse cytokinin synthesis.The results of Real-time PCR technique showed that the expression of DoIPT5a in sprouting bulbils was highest under the condition of water shortage at 22 ℃;and the expression of DoIPT1 and DoIPT5b genes was the highest in a humid environment;the expression of DOIPTs genes raised with paclobutrazol treatment.These results laid a foundation for further study on the function of DoIPTs in yam.

Key words: Yam, Gene cloning, DoIPT gene, Bulbil, Paclobutrazol(PP₃₃₃), Relative quantitative expression

唐文芳, 徐升胜, 段延碧, 龙雯虹, 杨荣萍, 张雪梅, 孟金贵. 山药DoIPTs基因克隆及珠芽发芽期表达分析[J]. 华北农学报, 2022, 37(1): 27-34. doi: 10.7668/hbnxb.20192488.

TANG Wenfang, XU Shengsheng, DUAN Yanbi, LONG Wenhong, YANG Rongping, ZHANG Xuemei, MENG Jingui. Cloning of DoIPTs Genes in Yam and Its Expression Analysis During Bulbil Germination[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 27-34. doi: 10.7668/hbnxb.20192488.

http://www.hbnxb.net/CN/Y2022/V37/I1/27

图/表 8

表1 PCR引物

Tab.1 Primers used for PCR

引物名称 Primer name	用途 Purpose	序列(5'—3') Primer sequence	扩增大小/bp Amplification length
DoIpt1	中间扩增	F: ATTCACTGTTGGTTCCCTTCTG	1 120
		R: AGGCTTCGACCTGCTTGTCT
	5'RACE	①:CGATGGACGGAGGAGTCTTGT	279
		②:GATGTCAAGCCCTCTATAAACCTG
	3'RACE	①:AGACGGCTGTGGCGGAGAT	56
		②:GAAGATCAAGCGATTGGTGGAG
	全长验证	F: GGCAAAGAGAAGCAGAAC	1 315
		R:GACAGCGATAACCTGACA
	qRT-PCR	F:TGCCGCATCATCTCCTCG	221
		R:ATACCTGAACCGCTCTCGC
DoIpt5a	中间扩增	F:ACCTCGCCCTCCACTTCA	1 044
		R:CACCGCCTTCATCATACA
	全长验证	F:GTGTCGCTATAATTGTTCT	1 026
		R:AGGTTGATAGCCCAAGATA
	qRT-PCR	F:GTTGAGGAAGCAAGAGGAGTG	237
		R:ATCCCACCCTGACTCCACC
DoIpt5b	中间扩增	F: CCCTTTCCACTTGAATCCC	1 108
		R: AATGGCTCCACGCACTAAC
	5'RACE	①:GAGAAGTGCTTGGCTAAGTCG	182
		②:GAATCTCGTCTATGGCTTTCAT
	3'RACE	①:CGAATGTTACTTCCTTTGGGTC	202
		②:ACACGCTGGCTTTGCTTC
	全长验证	F: GTGAGGGTGTAAGAGTCAA	1 315
		R:GGAGTTTAGAGCCAAGTATG
	qRT-PCR	F:GAATCCCACCACCTTCTCCA	178
		R:CTCGTCGGTGACTTTGTTGG
RACE	5'Outer	CATGGCTACATGCTGACAGCCTA
	5'Inner	CGCGGATCCACAGCCTACTGATGATCAGTCGATG
	3'RT	ACGACTCACTATAGGGCN
CHC	qRT-PCR	F: GGTGAACTGTATGAGGCGG	156
		R: GCATCCACACAAGCAAAGCA

图1 RNA、DoIPTs全长凝胶电泳图及DoIPTs基因的ORF编码区域

Fig.1 Gel electrophoresis map of RNA,DoIPTs full-length,and ORF encoding region of DoIPTs gene

图2 DoIPTs氨基酸的信号肽

Fig.2 Signal peptide of DoIPTs amino acids

图3 DoIPTs蛋白二级和三级结构预测模型
A:蓝色.α-螺旋;红色.延伸链;绿色.β-转角;橘色.无规卷曲。

Fig.3 Secondary structure and tertiary structure of DoIPTs proteins
A:Blue.α-helix;Red.Extended chain;Green.β-turn;Orange.Random curl.

图4 DoIPTs氨基酸序列比对
粉色显示同源级别为100%,蓝色同源级别为100%~75%,绿色高于75%~50%,白色低于33%;红色横线标识ATP/GTP结合位点GXXGXGK(X代表任意氨基酸)。

Fig.4 Sequence alignment of DoIPTs amino acids
Similarity of 100%,100%—75%,75%—50%,and 0—33% was displayed with pink,blue,green and white,respectively;The binding site GXXGXGK(X standed for any amino acid)of ATP/GTP was displayed with red horizontal line.

图5 DoIPTs氨基酸进化树
进化树枝叶由蛋白名及登录号代表,蛋白名前2个字母由物种拉丁名缩写得到。凤梨.AcIPT5;拟南芥.AtIPT(1—9)9个蛋白;深圳拟兰.AsIPT5;芜青.BrIPT3;油棕.EgIPT5;向日葵.HaIPT3;橡胶树.HbIPT3;木薯.MeIPT3;杨梅.MrIPT3;海枣.PdIPT5;可可.TcIPT3;野生二粒小麦.TdIPT5。本研究获得的蛋白用四菱形标出。

Fig.5 Evolutionary tree of amino acids sequence of DoIPTs
Branches and leaves in phylogenetic tree were represented by protein name and registration number,and the first two letters of the protein name were abbreviated from the Latin name of the species.Ananas comosus.AcIPT5;Arabidopsis thaliana.AtIPT(1—9)9 proteins;Apostasia shenzhenica.AsIPT5;Brassica rapa.BrIPT3;Elaeis guineensis.EgIPT5;Helianthus annuus.HaIPT3;Hevea brasiliensis.HbIPT3;Manihot esculenta.MeIPT3;Morella rubra.MrIPT3;Phoenix dactylifera.PdIPT5;Theobroma cacao.TcIPT3;Triticum dicoccoides.TdIPT5.The proteins obtained in this study were marked with four diamonds.

图6 DoIPTs在珠芽不同时期的相对表达量
图A为珠芽运用本体水分调节发芽时,珠芽表皮中基因的表达量;图B为珠芽采收60 d后,施加水后珠芽表皮中基因的表达量。图中不同小写字母表示差异显著(Duncan's法,P<0.05)。图7同。

Fig.6 Relative expression level of DoIPTs in bulbils at different periods
Fig.A shows the expression level of genes in the epidermis of bulbils when bulbils used their own water to regulate sprouting;Fig.B shows the gene expression level in the epidermis of bulbils after 60 days of bulbils harvest and application of water.Different lowercase letters indicate significant difference(Duncan's method,P<0.05).The same as Fig.7.

图7 DoIPTs在不同浓度多效唑处理后的相对表达量

Fig.7 Relative expression level of DoIPTs under different concentrations of PP₃₃₃

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