番木瓜干旱胁迫相关CpDHN1基因的克隆与分析  

Molecular Cloning and Analysis of CpDHN1,a Gene in Response to Drought Stress in Papaya(Carica papaya L.)

作　　者：郭静远[1] 邹智 孔华[1] 贺萍萍[1] 郭安平[1] GUO Jingyuan;ZOU Zhi;KONG Hua;HE Pingping;GUO Anping(Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions/Institute of Tropical Biosciences and Biotechnology/Sanya Research Institute of Chinese Academy of Tropical Agriculture Science,Sanya,Hainan 572024,China)

机构地区：[1]海南省南繁生物安全与分子育种重点实验室/中国热带农业科学院热带生物技术研究所/中国热带农业科学院三亚研究院,海南海口571101

出　　处：《热带作物学报》2021年第12期3492-3499,共8页Chinese Journal of Tropical Crops

基　　金：海南省自然科学基金项目(No.320MS103);海南省重大科技计划项目(No.ZDKJ202002);中国热带农业科学院基本科研业务费专项资金(No.1630052017015)。

摘　　要：番木瓜(Carica papaya L.)隶属于番木瓜科番木瓜属,是一种营养价值高的热带果树,广泛种植于热带和亚热带地区。作为全年结果的典型热带作物,番木瓜易受寒害、旱害等非生物胁迫的影响。然而,目前有关番木瓜的抗逆研究主要集中于导致绝产的病毒病,鲜见针对非生物胁迫的报道。脱水素又名第二类胚胎发育晚期丰富蛋白,属于亲水性高、富含甘氨酸、复杂度低的非结构蛋白,因其与生物的抗逆性密切相关而受到广泛关注。为解决番木瓜生产在地域上的局限性,并进一步提高其品质和产量,积极筛选和鉴定参与非生物胁迫响应的关键基因具有重要的理论意义和应用价值。本研究基于转录组获得的CpDHN1转录本序列,以‘台农二号’番木瓜组培苗叶片为材料,提取总RNA进行反转录,结合RT-PCR技术克隆了该基因636 bp的全长编码区序列,在此基础上对其进行序列、表达特性以及原核表达分析。结果显示,CpDHN1编码211个氨基酸,其理论分子量为24.09 kDa,等电点为5.05,总平均疏水指数为–1.584,不稳定系数为66.51,属于不稳定、高度亲水的细胞核定位蛋白。CpDHN1蛋白富含谷氨酸、赖氨酸和丝氨酸,含有3个保守区域,即2个K片段和1个S片段,符合脱水素蛋白的基本结构特征,属于SKn型脱水素。生物信息学分析表明CpDHN1无跨膜螺旋,其二级结构中以α-螺旋结构占主导。表达分析显示,该基因在根、叶片和韧皮部树液中均有表达,且其表达水平受干旱胁迫调控。同源分析表明,CpDHN1与拟南芥AtLEA4序列相似性最高,为46.8%,而与番木瓜中已报导的另一个脱水素CpDHN的相似性仅为19.4%。研究还构建了CpDHN1的原核表达载体,根据SDS-PAGE结果,CpDHN1在40 kDa处有条中强度的诱导条带,而在55 kDa处有条高亮的诱导条带,可能与其无规结构有关。这些结果为进一步的功能分析与应用奠定了坚实的基础。Papaya(Carica papaya L.),which belongs to the Carica genus within the Caricaceae family,is a popular tropical fruit with high nutritional value that are widely cultivated in tropical and subtropical areas.As a typical tropical crop that fruits through the whole year,papaya is vulnerable to abiotic stresses such as cold and drought.Presently,there are few reports on stress resistance of papaya,and main studies focus on the virus disease which leads to the extinction of papaya production.Dehydrin,also known as type two late embryogenesis abundant protein,is a class of natural non-structural proteins with high hydrophilicity,rich in glycine,and low complexity.It has attracted extensive attention because it is highly related to the ability of plants to resist abiotic stresses.In order to solve the regional limitation of papaya cultivatation and further improve the fruit quality and yield,it is of importance to screen and characterize key genes involved in abiotic stress response.Based on a de novo assembled transcript,a 636 bp open reading frame of a gene that was denoted as CpDHN1 was isolated from the leaf tissue of cultivar‘Tainong No.2’using RT-PCR.Bioinformatic analysis showed that CpDHN1 putatively encodes 211 amino acids,which was predicted to target the nuclear and have a theoretical molecular weight(Mw)of 24.09 kDa,an isolectric point(pI)of 5.05,a GRAVY value of–1.584,and an instability index of 66.51,implying its unstable,hydrophilic,and nuclear-targeted characteristics.CpDHN1 is rich in glutamate,lysine and serine,and it can be categorized as the SKn-type dehydrin with two conserved K-segments and one S-segment.CpDHN1 doesn’t contain any transmembrane helix and its secondary structure is mainly composed ofα-helixes.Homology analysis showed that CpDHN1 shares the highest sequence similarity with AtLEA4(i.e.46.8%),while the sequence similarity with another reported dehydrin(i.e.CpDHN)is just 19.4%.Expression analysis revealed that CpDHN1 was expressed in all tested tissues,i.e.root,leaf,and sap,and

关 键 词：番木瓜 胚胎发育晚期丰富蛋白 脱水素 干旱胁迫 原核表达 

分 类 号：S667.9[农业科学—果树学]