摘 要:【目的】探究陆地棉C2H2型锌指蛋白基因GhZFP8在棉花抗黄萎病反应中的功能,为挖掘棉花抗病基因奠定理论基础。
【方法】通过同源克隆方法得到一个陆地棉C2H2型锌指蛋白基因GhZFP8,利用生物信息学方法分析该基因的理化性质,构建该基因病毒诱导的基因沉默(VIGS)载体,通过农杆菌介导法转化棉花,检验GhZFP8的抗病功能。
【结果】GhZFP8开放阅读框(ORF)为789 bp、编码为262个氨基酸,相对分子量为28.12 kD、等电点为8.16、脂肪指数为59.31和平均疏水性为-0.718,为亲水性、碱性的非跨膜蛋白。GhZFP8蛋白具有2个ZnF-C2H2结构域,含有6个α-螺旋和4个β-折叠,属于C2H2型锌指蛋白。GhZFP8基因沉默植株对黄萎病菌的抗性相对减弱。
【结论】GhZFP8基因在棉花抗黄萎病过程中发挥正调控作用。
关键词:黄萎病;GhZAT8;基因克隆;病毒诱导的基因沉默
中图分类号:S562 ""文献标志码:A
文章编号:1001-4330(2025)01-0137-09
收稿日期(Received):
2024-08-11
基金项目:
国家自然科学基金项目(32160494);新疆维吾尔自治区自然科学基金项目(2023D01E03);国家级大学生创新创业训练计划项目(202210758001);新疆维吾尔自治区重点研发专项(2022B02033-1)
作者简介:
程贯富(1998-),男,山东济南人,硕士研究生,研究方向为棉花逆境分子生物学,(E-mail) cgfyouxi@163.com
通信作者:
李月(1984-),女,河南许昌人,副教授,博士,研究方向为棉花逆境分子生物学,(E-mail) liyue6905@126.com
0 引 言
【研究意义】棉花黄萎病是由大丽轮枝菌引起的,严重为害棉花生产[1]。目前尚缺乏行之有效的杀菌剂,因此培育抗病品种成为防控棉花黄萎病的有效措施[2],传统杂交育种方法培育抗病品种难度大,周期长[3-4]。因此利用分子生物学方法挖掘抗病基因,对培育棉花抗病品种、有效防控棉花黄萎病有重要意义。【前人研究进展】植物抗病过程涉及多种基因及信号传递途径,例如转录因子调控、蛋白激酶、激素信号途径、防卫基因合成等。其中转录因子在植物抗病过程中发挥重要作用,已成为研究植物抗病育种的关键因素。Zhang等[5]发现玉米JAZ家族转录因子ZmJAZ在茎腐病和炭疽病菌侵染植株后,其表达量发生变化。Yu等[6]将葡萄TIFY家族转录因子基因VvTIFY9过表达后,发现植株对白粉病的抗性增强。Zang等[7]研究表明,玉米转录因子ZmERF105能增强植株对大斑病菌的抗性。锌指蛋白(zinc finger proteins, ZFPs)是真核生物基因组中最丰富的转录因子之一,ZFPs具有高度保守的结构域,由大约20~30个氨基酸组成,共识序列为CX2-4CX3FX5LX2HX3-5H(X代表任何氨基酸,下标:氨基酸数)[8],Berg等[9]将其分为9类,分别为C4、C6、C8、CCCH、C2HC、C2HC5、C2H2、C3HC4和C4HC3,其参与调节植物多种生长发育过程,抵抗生物和非生物胁迫[10]。Mittler等[11]发现拟南芥Zat10基因表达可以快速适应外界温度的变化。Davletova等[12]又发现在多种非生物胁迫的信号传导途径中均检验到Zat12蛋白。Kim等[13]又发现大豆SCOF-1基因受到低温和ABA胁迫后表达上调,增强了植株的耐冻性。已经报道了许多参与植物抗病的ZFPs基因,Tian等[14]发现在马铃薯受到致病菌感染后StZFP1基因表达量增加,并提高了植株的抗病性。Uehara等[15]通过试验发现 NtZFT1基因响应烟草花叶病毒的入侵,并增强了对烟草花叶病毒的抗性。Li等[16]在水稻抗稻瘟病品种“地谷”中发现一个编码锌指蛋白的基因bsr-d1,其能调控H2O2酶基因表达,影响水稻对稻瘟病的抗性。其中王亚等[17]将Bsr-d1基因启动子区域的一个关键碱基突变,导致MYB转录因子与Bsr-d1启动子结合增强,抑制Bsr-d1基因表达,使细胞内H2O2富集,提高了水稻的免疫反应并因此获得稻瘟病抗性。近几年报道了许多参与防御棉花黄萎病的基因,如LYK1和LYK2[18],GbCYP86A1[19],GhWAK7A [20]和GhWAKL[21]。【本研究切入点】锌指蛋白在棉花抗黄萎病中发挥作用的报道相对较少。前期通过同源克隆方法,在陆地棉基因库中找到并克隆水稻抗稻瘟病基因Bsr-d1的同源基因,并命名为GhZFP8,需通过结构预测其是否具有C2H2型锌指蛋白特征。【拟解决的关键问题】采用生物信息学的方法分析其理化性质,利用病毒诱导基因沉默(virusinduced gene silencing,VIGS)技术,验证其是否在棉花黄萎病抗性过程中发挥作用,研究该基因抗病机制提供理论基础,为棉花抗病品种的培育提供科学依据。
1 材料与方法
1.1 材 料
供试植物材料中棉35、农杆菌菌株为GV3101、棉花黄萎病菌菌株V991、TRV病毒载体及含阳性对照TRV:GhCLA1载体的农杆菌菌株,由新疆农业大学生命科学学院作物功能基因组学与分子改良实验室保存;卡那霉素、庆大霉素、MES、乙酰丁香酮、MgCl2及培养基均为国产分析纯试剂;多糖多酚植物总RNA提取试剂盒购于杭州博日科技公司;EcoRI和KpnI等限制性内切酶购于赛默飞(Thermo)公司, TaqDNA聚合酶、T4 DNALigase、RNaseA、高保真聚合酶TransStar KD Plus、反转录试剂盒、荧光定量试剂盒、琼脂糖凝胶回收试剂盒和DNA分子量Marker均购自北京全式金生物技术有限公司。PCR所用引物的合成及DNA的测序均由上海生工生物工程有限公司完成。
1.2 方 法
1.2.1 种植及培养
陆地棉中棉35种子经硫酸脱绒后,选取大小一致的种子,利用胡子曜等[22]方法种植。
1.2.2 总RNA的提取及cDNA的合成
参照RNA提取试剂的说明书提取棉花叶和根部样品的总RNA。经琼脂糖凝胶电泳检验后,用反转录试剂盒合成cDNA。
1.2.3 GhZFP8基因的克隆及序列分析
通过对水稻Bsr-d1的氨基酸序列在陆地棉数据库中进行Blastp比对,获得棉花基因GhZFP8序列,其NCBI(https://www.ncbi.nlm.nih.gov/)的登录号为XM_016850242,利用表1工具,进行GhZFP8基因生物信息学分析。通过其CDS序列信息设计特异性引物,参照李秀青等[23]的方法,以中棉35的cDNA为模板,进行PCR扩增,扩增产物经琼脂糖凝胶电泳检测后,回收目的条带,并连接pEASY Blunt-Zero克隆载体,转化大肠杆菌Trans-T1感受态细胞,挑取阳性单克隆质粒进行测序。表1,表2
1.2.4 GhZFP8基因的VIGS载体构建
利用SGN-VIGS网站(https://vigs.solgenomics.net/)设计GhZFP8 基因的沉默片段序列,沉默片段大小为318 bp,利用DNAMAN6软件设计该序列的上下游的引物,利用SnapGemne软件在2个引物的5′端分别加入EcoRI和 KpnI的酶切位点。以棉花叶片cDNA为模板,用PCR技术扩增沉默片段序列,将PCR产物中符合大小的目的片段回收连接克隆载体B-zero载体并测序,参考胡子曜等[22]方法,将测序正确的目的片段与TRV2线性化载体用T4 DNA Ligase连接,得到GhZFP8基因的VIGS沉默载体,将质粒转化DH5α大肠感受态细胞,扩大培养提取质粒并双酶切验证,将验证正确的质粒通过冻融法转化到GV3101 感受态细胞中,28℃培养2~3 d以备下一步的试验。表2
1.2.5 农杆菌介导的VIGS侵染棉花及沉默效率检测
将载体TRV:RNA2、TRV:GhCLA1、TRV:GhZFP8和TRV:RNA 1分别转化农杆菌,待棉花苗生长至2片子叶完全展开时,选取生长较为一致的棉花幼苗,参照Hou等[24]方法,进行VIGS侵染棉花。分别将3组混合重悬菌液(TRV:GhCLA1和TRV:RNA1、TRV:RNA2和TRV:RNA1、TRV:GhZFP8和TRV:RNA1)侵染的棉花植株作为阳性、阴性对照及试验组。当阳性对照植株TRV:GhCLA1的叶片出现明显的白化表型时,于试验组和阴性、阳性对照的第2片真叶及根部取样,提取RNA,反转cDNA,按照荧光定量试剂盒说明书进行qRT-PCR反应,检测目的基因沉默效率。
以棉花管家基因GhUBQ7[25]为内参基因,进行3个技术重复。反应条件与体系按照王倩等[26]方法进行,根据目的基因和内参基因Ct值,使用2-ΔΔCt方法[16]计算目的基因的表达量。表2
1.2.6 GhZFP8黄萎病抗性的鉴定
参考Li等[27]方法对沉默植株进行黄萎病接种处理,15 d后观察沉默植株与对照植株的表型差异,采用叶片分级法统计病情指数[27],对沉默植株和对照植株进行剖杆检测[28],并对接种48 h的沉默植株进行植物木质化检测[28]。
2 结果与分析
2.1 GhZFP8基因的克隆与序列
研究表明,GhZFP8基因克隆成,其开放阅读框(ORF)为789 bp,编码一个含262个氨基酸,相对分子量为28.12 kD,等电点为8.16的碱性氨基酸,脂肪指数为59.31,平均疏水性为-0.718的亲水蛋白。GhZFP8蛋白有99.93%的概率不存在信号肽,预测GhZFP8属于非跨膜蛋白。GhZFP8蛋白的结构域预测具有2个ZnF-C2H2结构域。其二级结构预测可知,GhZFP8蛋白含有6个α-螺旋和4个β-折叠,其三级结构预测符合二级结构的所预测的特征,其符合锌指蛋白的特征。图1~4
2.2 "GhZFP8基因的VIGS载体构建
研究表明,将抑制GhZFP8表达的靶序列用PCR的方法扩增,目的片段大小为318 bp符合预期。所扩增的片段与目的片段一致。随后将其与VIGS沉默载体TRV2连接,随后用EcoRI和 KpnI进行酶切验证,所得结果与预期结果一致,为一条大于8 000 bp的条带和一条在250~500 bp的目的片段条带。GhZFP8的VIGS载体构建成功。图5
2.3 GhZFP8的沉默效果检测
研究表明,将TRV:RNA2、TRV:GhCLA1、TRV:GhZFP8农杆菌分别与TRV:RNA1农杆菌等比例混合,分别侵染棉花子叶。15 d后表型观察,侵染TRV﹕GhCLA1的阳性对照植株出现白化现象,利用qRT-PCR技术检测阳性对照植株中根和真叶的GhCLA1表达量。阳性对照植株中GhCLA1的表达量低于对照组,VIGS载体能够在植株体内正常工作。同时利用qRT-PCR技术检测目的基因GhZFP8在根和真叶的表达量,与对照组相比,沉默植株的GhZFP8的表达量明显低于对照组,获得GhZFP8基因沉默植株。图6
2.4 GhZFP8基因沉默植株的黄萎病抗性鉴定
研究表明,与对照植株相比,TRV:GhZFP8基因沉默植株出现叶片边缘发黄,萎蔫症状。TRV:00对照植株病情指数统计为37,GhZFP8基因沉默植株病情指数为43%,显著高于对照植株。各植株茎秆均不同程度发生褐变,TRV:GhZFP8植株褐变程度比对照组较深。取接种黄萎病菌后48 h的棉花茎段切片放置于体式显微镜下观测植株木质化程度,TRV:GhZFP8沉默植株木质化程度较弱。GhZFP8基因对抵御黄萎病的侵染可能具有积极作用。图7
3 讨 论
3.1
棉花抗黄萎病反应的过程需要一系列相关蛋白的参与,随着棉花基因组测序的完成,一系列的抗黄萎病病基因陆续被挖掘鉴定:Dong等[29]从雷蒙德氏棉克隆一个ATP结合蛋白基因ABCF5,并通过VIGS技术验证了其是棉花抗黄萎病反应的负调控因子。Qin等[30]发现棉花GhCyP3基因可以抑制U-box E3泛素连接酶基因GhPUB17的表达,使其在棉花抗黄萎病反应中发挥负调控作用。Han等[31]在棉花中克隆了棉花宿主细胞分泌几丁质酶基因Chi28和富含半胱氨酸的重复蛋白基因CRR1并且又在大丽轮枝菌中克隆得到分泌型丝氨酸蛋白酶基因VdSSEP1,通过试验表明VdSSEP1蛋白可以分解Chi28蛋白,CRR1蛋白保护Chi28蛋白不被VdSSEP1分解,而将CRR1和Chi28基因敲除后,植株突变体易感棉花黄萎病菌,反之将大丽轮枝菌VdSSEP1基因沉默后,则破坏了大丽轮枝菌的致病性。Gong等[32]在215份接种黄萎病菌的中国棉属材料进行全基因研究,鉴定出一种GST基因GaGSTF9,其可能通过SA相关信号通路影响棉花黄萎病菌的抗性,并通过试验验证该基因正调控棉花黄萎病抗性。He等[33]在棉花中鉴定出一个可以抑制JA应答基因表达的HD-ZIPⅠ类转录因子基因GhHB12,发现其对棉花抗黄萎病反应进行负向调控。
3.2 C2H2型锌指蛋白是生物体内普遍存在的一类转录因子,有研究表明其在植株受到生物胁迫时发挥作用。Zhang等[34]在烟草中发现C2H2型锌指蛋白NbcZF1可以通过ROS-NO途径介导多种SsCut(Sccutotinia sclerotiorum cutinase是一类诱导子可以诱导植物免疫)触发的反应,增强烟草对烟草黑胫病等疾病抗性。Noman等[35]在辣椒中发现C2H2型CaZNF基因沉默后,植株对青枯病的抗性降低。Shi等[36]在拟南芥鉴定出C2H2型锌指蛋白AtZAT6通过与防御基因的启动子相结合正向调控植株体内SA与ROS积累,从而提高植株对丁香假单胞杆菌的抗性。目前有关C2H2型锌指蛋白在棉花抗黄萎病中发挥作用的报道相对较少。
3.3 研究通过同源对比方法克隆得到一个C2H2型锌指蛋白基因GhZFP8,该基因开放阅读框(ORF)为789 bp,编码一个含262个氨基酸,相对分子量为28.12 kD,等电点为8.16的碱性氨基酸。GhZFP8蛋白脂肪指数为59.31,平均疏水性为-0.718的亲水蛋白。GhZFP8蛋白有99.93%的概率不存在信号肽,预测GhZFP8属于非跨膜蛋白。GhZFP8蛋白的结构域预测具有2个ZnF-C2H2结构域。其二级结构预测可知,GhZFP8蛋白含有6个α-螺旋和4个β-折叠,其三级结构预测符合二级结构的所预测的特征。通过VIGS技术得到GhZFP8沉默植株后并接种黄萎病菌,与对照植株相比,GhZFP8沉默植株出现叶片边缘发黄和萎蔫状态,测得沉默植株的病情指数为43%,高于对照植株。通过纵剖茎秆观察,发现沉默植株褐变程度比对照组较深,将植株进行木质部染色,观察沉默植株木质部比对照植株较薄但差异较小。推测GhZFP8在棉花抗黄萎病过程中可能参与抗病过程,其在棉花抗黄萎病反应中具体发挥的作用需进一步试验验证。
4 结 论
克隆获得C2H2型锌指蛋白基因GhZFP8,并初步探究其在棉花中的抗病功能,其开放阅读框(ORF)为789 bp,编码一个含262个氨基酸,相对分子量为28.12 kD,等电点为8.16的碱性氨基酸,GhZFP8蛋白脂肪指数为59.31,平均疏水性为-0.718的亲水蛋白。GhZFP8蛋白有99.93%的概率不存在信号肽,预测GhZFP8属于非跨膜蛋白,GhZFP8蛋白的结构域预测具有2个ZnF-C2H2结构域。GhZFP8蛋白含有6个α-螺旋和4个β-折叠。VIGS技术抑制该基因表达后,其病情指数略高于阴性对照植株;基因沉默后棉花对黄萎病的抗性相对减弱,推测GhZFP8在棉花抗黄萎病过程中发挥正调控作用。
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Functional analysis of a C2H2 zinc protein GhZFP8
gene in Gossypium hirsutum resistance to Verticillium wilt
CHENG Guanfu,LU Guoqiang,CUI Yongxiang,HOU Aocheng,ZHANG Guoshuai,
LIANG Chunyan,LEI Jianfeng,LU Wei,DAI Peihong,LI yue
(Xinjiang Key Laboratory for Ecological Adaptation and Evolution of Extreme Environment Biology/College of Life Sciences, Xinjiang Agricultural University, Urumqi 830052, China)
Abstract:【Objective】 To explore the function of the terrestrial cotton C2H2-type zinc finger protein gene GhZFP8 in the response to Verticillium wilt in cotton in the hope of laying a theoretical foundation for mining cotton disease resistance genes.
【Methods】 The cotton gene GhZFP8 was obtained by homologous cloning and its biological function explored. Bioinformatics methods were used to analyze the physicochemical properties of the gene, while the VIGS vector for the gene was constructed and transformed into cotton by Agrobacterium-mediated method to preliminarily test the disease resistance function of GhZFP8.
【Results】 The open reading frame (ORF) of GhZFP8 was 789 bp, encoded as a hydrophilic protein containing 262 amino acids, a relative molecular weight was 28.12 kD, a basic amino acid with an isoelectric point of 8.16, a lipid index of 59.31, and an average hydrophobicity of-0.718. The absence of a signal peptide in the GhZFP8 protein predicted that GhZFP8 was a non-transmembrane protein. GhZFP8 protein revealed that it had 2 ZnF-C2H2 structural domains. Its secondary structure prediction revealed that the GhZFP8 protein contained 6 α-helices and 4 β-folds. GhZFP8 gene silenced plants showed relatively reduced resistance to Verticillium wilt.
【Conclusion】 The GhZFP8 gene plays a positive regulatory role in cotton resistance to verticillium wilt.
Key words:Verticillium wilt;GhZFP8; gene cloning;VIGS
Fund projects:" National Natural Science Foundation of China (32160494); Project of Natural Science Foundation of Xinjiang Uygur Autonomous Region (2023D01E03);" Project of National Innovation and Entrepreneurship Training Program for College Students (202210758001);" Key Scientific R amp; D Program Project of Xinjiang Uygur Autonomous Region (2022B02033-1)
Correspondence author: LI Yue (1984-), female, from Xuchang, Henan, doctor, associate professor, research direction: molecular biology of cotton adversity, (E-mail) liyue6905@126.com