毛竹PeSCR基因的表达与功能

doi:10.11707/j.1001-7488.20160605

摘要/Abstract

摘要： [目的] SCARECROW(SCR)基因在植物根和茎顶端细胞不均等分裂形成基本组织的过程中发挥着重要调控作用。通过分析毛竹中SCR同源基因PeSCR的结构特点,研究该基因的组织表达特异性,分析激素GA₃、ABA以及干旱、NaCl等非生物胁迫处理对该基因的表达影响,利用在拟南芥中过量表达PeSCR,初步鉴定其功能,以期为竹子分子育种提供基因资源。[方法] 采用生物信息学的方法,在毛竹数据库(BambooGDB)中获得SCR同源基因序列和上游调控序列,分别利用Spidey和Plant CARE在线软件分析基因结构特点及其上游调控序列所含作用元件,采用实时荧光定量PCR技术分析基因在不同组织中的表达性,以及GA₃、ABA、干旱和NaCl等非生物胁迫处理后的表达变化,构建PeSCR基因的正义/反义表达载体,转化拟南芥,通过分析转基因植株的表型来判断基因的功能。[结果] 从毛竹中获得SCR同源基因PeSCR(登录号:FP094510),cDNA全长为2301 bp,其中5'和3'端非编码区分别为238,134 bp,编码区1929 bp。编码区对应的基因组序列为2598 bp,包含1个内含子(672 bp)。PeSCR编码1个642个氨基酸的蛋白,该蛋白具有GRAS家族的典型结构域(LRⅠ,VHIID,LRⅡ,PFYRE和SAW),属于AtSCR亚家族。PeSCR蛋白与其他植物SCR有很高的同源性,其中与水稻的OsSCR2和拟南芥的AtSCR的一致性分别为84.9%,54.9%。PeSCR上游调控序列为1820 bp,包含生长素应答元件AuxRR-core、ABA应答元件Motif Ⅱb、干旱诱导MYB结合位点MBS、光应答元件等多种作用元件,这意味着PeSCR可能受到激素、干旱等的调控。qPCR结果表明,PeSCR在叶中的表达丰度最高,其次是根和茎,而鞘中最低;PeSCR的表达短时间内受GA₃的抑制,随处理时间延长(至5 h),基因的表达受到诱导;PeSCR的表达总体受外源ABA和NaCl处理的抑制;干旱处理条件下PeSCR基因表达呈先上升后下降的趋势。RT-PCR证明PeSCR已在转基因拟南芥植株中得到表达,表型分析发现,与野生型相比转正义基因植株生长健壮,根系发达,而反义植株矮小,根系生长受到抑制。[结论] 在毛竹各组织中PeSCR呈组成型表达,根中表达受到GA₃、ABA以及干旱、NaCl的影响。该基因正义表达促进转基因植株生长,反义转基因植株则受到抑制,表明该基因可能参与毛竹的生长发育调控。

关键词: 毛竹, PeSCR, 激素, 非生物胁迫, 基因表达, 功能分析

Abstract: [Objective] The SCARECROW(SCR) gene played important roles in asymmetric cell divisions to generate basic tissues in the root and shoot of plants. The structural features of SCR homologous gene PeSCR in Phyllostachys edulis were analyzed, the tissue specific expression of PeSCR was studied, the influence on PeSCR expression in roots treated with GA₃, ABA, drought and NaCl was investigated respectively, and the function was preliminarily identified by overexpressing PeSCR in Arabidopsis thaliana, which would be helpful to reveal the function of PeSCR and provide genetic resources for molecular breeding of bamboo.[Method] The sequence of SCR homologue gene PeSCR and its upstream regulatory sequence in P. edulis were retrieved from the bamboo database (BambooGDB) using bioinformatics methods. The gene structure was analyzed with Spidey and the acting elements in upstream regulatory sequence were detected using Plant CARE online softwares respectively. The tissue specific expression of PeSCR in different tissues, as well as the changes of PeSCR expression in roots treated with GA₃, ABA, drought and NaCl were analyzed using real-time quantitative PCR (qPCR). The expression vectors of sense and antisense PeSCR were constructed and transformed into A. thaliana, and the phenotype of transgenic plants was investigated to identify the function of PeSCR. [Result] A SCR homologous gene, PeSCR (GenBank No. FP094510) was obtained, the full length of cDNA was 2301 bp, including a 5' untranslated region (UTR) of 238 bp, a 3' UTR of 134 bp and an open reading frame (ORF) of 1929 bp. The genomic sequence corresponding to ORF of PeSCR was 2598 bp containing one intron of 672 bp. PeSCR encoded a protein with 642 amino acids, which had typical domains (LRⅠ, VHIID, LRⅡ, PFYRE and SAW) of GRAS family and belonged to AtSCR subfamily. PeSCR had high homology with SCRs from other monocots, among which the identities with OsSCR2 in rice and AtSCR in A. thaliana were 84.9% and 54.9%, respectively. The regulatory sequence upstream of PeSCR was 1820 bp, including many kinds of responsive elements such as cis-acting regulatory element (AuxRR-core) involved in auxin responsiveness, abscisic acid responsive element (Motif Ⅱb), MYB binding site (MBS) involved in drought-inducibility and light responsive elements, indicating that PeSCR might be regulated by hormones, drought, etc. The qPCR analysis indicated that PeSCR expressed constitutively at a high level in leaf, followed by root and stem, and the least was in sheath. The expression of PeSCR in root was inhibited by GA₃ in short time (within 1 h), but it was induced with prolonged treatment (up to 5 h). Overall, PeSCR was suppressed by exogenous ABA and NaCl, while it was induced initially and subsequently suppressed by drought. The expression of PeSCR in transgenic plants of A. thaliana was confirmed by RT-PCR. The phenotypes demonstrated that sense transgenic plants had a vigorous growth and well-developed root system compared to the wild type, while antisense transgenic plants were small and the growth of root was restrained. [Conclusion] The results indicated that the expression of PeSCR was constitutive in different tissues, and influenced by the GA₃, AAB, drought and NaCl in roots. The expression of sense PeSCR promoted the growth of transgenic plants, while that of antisense inhibited plant growth, suggesting that PeSCR might participate in regulating process of P. edulis growth and development.

Key words: Phyllostachys edulis, PeSCR, hormone, abiotic stress, gene expression, functional analysis

中图分类号:

S718.46

董丽莉, 赵韩生, 王丽丽, 孙化雨, 娄永峰, 高志民. 毛竹PeSCR基因的表达与功能[J]. 林业科学, 2016, 52(6): 35-42.

Dong Lili, Zhao Hansheng, Wang Lili, Sun Huayu, Lou Yongfeng, Gao Zhimin. Expression and Function of PeSCR Gene from Phyllostachys edulis[J]. Scientia Silvae Sinicae, 2016, 52(6): 35-42.

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