橡胶树转录因子HbERF1的克隆与功能分析

doi:10.3864/j.issn.0578-1752.2014.08.018

摘要/Abstract

摘要： 【目的】克隆橡胶树转录因子HbERF1，分析其在橡胶树中响应非生物胁迫的表达模式，并通过在拟南芥中过表达鉴定其生物学功能，为橡胶树抗逆育种提供基因储备。【方法】利用RACE技术从巴西橡胶树中克隆到HbERF1全长序列，通过qRT-PCR技术分析其在非生物胁迫下的时空表达特性；通过农杆菌介导法转化拟南芥，利用Southern杂交和qRT-PCR技术对过表达植株进行鉴定，并分析过表达植株在干旱、高盐和PEG等胁迫条件下的表型及相关基因的表达特性，验证HbERF1的生物学功能。【结果】橡胶树转录因子HbERF1没有内含子，GenBank登录号为JQ914647，cDNA序列全长为1 178 bp，包括62 bp的5'非编码区、474 bp的3'非编码区和642 bp的开放阅读框。开放阅读框编码一条213个氨基酸组成的多肽，该蛋白具有一个保守的AP2结构域和一个EAR基序。系统进化分析表明，HbERF1属于AP2/ERF家族中ERF亚族的B-1类，与蓖麻RcERF、杨树PtERF46、拟南芥AtERF4的一致性最高，分别为72%、62%和61%。qRT-PCR分析表明，HbERF1在橡胶树叶片和树皮中均能响应高盐、干旱和PEG胁迫，基因的表达在叶片中受ABA、MeJA的抑制，而在树皮中则受ABA、MeJA和SA的抑制。在高盐和PEG胁迫下，叶片中的HbERF1在处理前期（≤8 h）表达水平都低于对照，而树皮中的HbERF1在胁迫4 h时就受到强烈诱导。在干旱胁迫下，随着干旱程度的增加，HbERF1在叶片和树皮中表达水平持续增加，且在树皮中的表达要高于叶片中的表达。HbERF1的过表达提高了拟南芥的抗旱性、耐盐性和耐PEG胁迫的能力，并抑制了乙烯受体基因AtETR1和AtERS1的表达。在过表达植株中，AtETR1和AtERS1的表达水平分别比野生型中的降低了19倍和9倍，差异极显著（P＜0.01）。在高盐胁迫下，过表达植株中AtRD22和AtRD29A的表达水平持续上升，且上调幅度高于WT，分别为对照的115倍和100倍；在20% PEG胁迫下，AtRD22和AtRD29A的表达都受到诱导。用300 mmol•L-1NaCl浇灌拟南芥，处理15 d后，野生型拟南芥的叶片几乎全部萎蔫白化，而过表达株系S1仅有少数叶片萎蔫白化。用20% PEG6000水溶液浇灌拟南芥，处理15 d后，过表达株系S1生长正常，已进入生殖生长，而野生型植株仍保持在营养生长阶段，生殖生长被延迟，且部分叶片出现脱水现象。停止浇水2周后，野生型拟南芥植株叶片枯萎程度高于过表达植株，复水1周后野生型植株的存活率仅为35.0%，而过表达植株的存活率则高达95.0%。【结论】HbERF1参与了橡胶树的ABA、JA、SA和ETH信号途径，是ETH信号的正调控因子，对提高橡胶树的耐旱性具有积极作用。

关键词: 巴西橡胶树 , 转录因子 , 非生物胁迫 , ERF

Abstract: 【Objective】AP2/ERF transcription factors play important roles in response to abiotic stress in plant. The objectives of this study are to clone ERF genes related to abiotic stress in rubber tree, analyze their expression patterns in response to abiotic stress and identify their biological functions by overexpressing in Arabidopsis thaliana so that it can provide candidate genes for rubber breeding in relation to stress tolerance. 【Method】The full cDNA sequence of HbERF1 was cloned from the rubber tree by RACE technology, then the temporal and spatial expression patterns of this gene were investigated under abiotic stress by real-time PCR. The HbERF1-overexpressing Arabidopsis was established by Agrobacterium-mediated transformation. The overexpressing lines were identified by Southern blotting and qRT-PCR. The phenotype and gene expression were analyzed under drought, NaCl and PEG stress in overexpressing lines, and the functions of HbERF1 were characterized preliminarily. 【Result】 HbERF1 (GenBank No. JQ914647) had no intron. The full-length cDNA of HbERF1 was 1 178bp in length, containing a 642-bp open reading frame flanked by a 62-bp 5’-UTR and a 474-bp 3’-UTR. HbERF1 encoded a 213 aa protein, which contained one AP2 domain and one EAR motif. The results of phylogenetic analysis showed that HbERF1 protein was classed into B-1 subgroup of ERF subfamily, and it showed 72%, 62%, and 61% identity with RcERF, PtERF46 and AtERF4, respectively. The real-time PCR analysis showed that HbERF1 was NaCl-, PEG- and drought -responsive in the leaves and bark of rubber tree. The expression of HbERF1 was repressed by abscisic acid (ABA) and methyl jasmonate (MeJA) in leaves and bark, meanwhile also by salicylic acid (SA) in the bark. Under NaCl- and PEG stress, HbERF1 was down-regulated in the first stress phase (≤8 h) in leaves, while HbERF1 was strongly induced in bark when it was stressed for 4h. Under drought stress, the expression of HbERF1 continuously increased in leaves and bark with the increase of drought degree, and the expression was higher in bark than in leaves. The overexpression of HbERF1 enhanced the tolerance to NaCl, PEG and drought in Arabidopsis. The expression of ethylene receptor gene, AtETR1 and AtERS1, were also repressed by HbERF1 overexpression in Arabidopsis, in which the expression of AtETR1 and AtERS1 was about 19-fold and 9-fold lower than those in the control, respectively, and the difference was highly significant(P＜0.01). Under NaCl stress, the expression of AtRD22 and AtRD29A had a 115-fold and 100-fold increase in overexpressing Arabidopsis, respectively, and these two genes were also induced under 20% PEG stress. Nearly all of the leaves became dehydrated and wilted in wild-type Arabidopsis after it was watered with 300 mmol•L-1NaCl for 15 days, while only few leaves became dehydrated and wilted in overexpressing line S1. Line S1 had been in the reproductive growth since it was watered with 20% PEG for 15days, while wild-type Arabidopsis was still in the vegetative growth, and some leaves became dehydrated. After stopping watering for 2 weeks, wild-type Arabidopsis was more wilted than line S1; and the survival rate of wild-type and overexpressing Arabidopsis was 35.0% and 95.0% after re-watered for 1 week, respectively. 【Conclusion】 HbERF1 was induced by ABA, MeJA, SA and ethylene (ETH), and it is a positive regulator of ETH-responsive genes and drought tolerance in the rubber tree.

Key words: Hevea brasiliensis , transcription factor , abiotic stress , ERF

安泽伟1, 谢黎黎1, 2, 王其同1, 李雅超1, 程汉1, 胡彦师1, 黄华孙1. 橡胶树转录因子HbERF1的克隆与功能分析[J]. 中国农业科学, 2014, 47(8): 1622-1633.

AN Ze-Wei-1, XIE Li-Li-1, 2 , WANG Qi-Tong-1, LI Ya-Chao-1, CHENG Han-1, HU Yan-Shi-1, HUANG Hua-Sun-1. Molecular Cloning and Functional Characterization of Transcription Factor HbERF1 in Hevea brasiliensis[J]. Scientia Agricultura Sinica, 2014, 47(8): 1622-1633.

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