中国农业科学 ›› 2018, Vol. 51 ›› Issue (23): 4409-4423.doi: 10.3864/j.issn.0578-1752.2018.23.002
张旭(),凌辉,刘峰,黄宁,王玲,毛花英,李聪娜,汤翰臣,苏炜华,苏亚春,阙友雄(
)
收稿日期:
2018-06-12
接受日期:
2018-07-29
出版日期:
2018-12-01
发布日期:
2018-12-12
基金资助:
ZHANG Xu(),LING Hui,LIU Feng,HUANG Ning,WANG Ling,MAO HuaYing,LI CongNa,TANG HanChen,SU WeiHua,SU YaChun,QUE YouXiong(
)
Received:
2018-06-12
Accepted:
2018-07-29
Online:
2018-12-01
Published:
2018-12-12
摘要:
【目的】WRKY是植物特有的一类转录因子,在生理调控和逆境响应过程中有着重要作用。通过分析WRKY转录因子基因在甘蔗生长发育及抗逆境过程中的作用,为甘蔗抗逆分子育种提供优良的基因资源。【方法】从甘蔗转录组数据库中挖掘到一条WRKY基因Unigene序列,并通过RT-PCR扩增得到cDNA全长序列,利用ORF finder、Smart、ExPaSy、Prabi、NetPhos、Cell-PLOC 2.0和DNAMAN6.0软件对该基因序列及其编码蛋白序列进行生物信息学分析,并使用MEGA6.0软件构建系统进化树;构建pCAMBIA1300-WRKY-GFP融合表达载体,通过农杆菌转化本氏烟(Nicotiana benthamiana),确定WRKY蛋白在烟草叶片中的亚细胞定位;利用酵母杂交试验验证WRKY是否具有转录自激活活性;采用实时荧光定量PCR(qRT-PCR)方法分析WRKY在甘蔗品种ROC22中的组织特异性(根、蔗芽、叶、蔗髓和皮)及其在MeJA(100 μmol·L -1)、SA(5 mmol·L -1)、PEG(25%)、NaCl(250 mmol·L -1)、CuCl2(500 mmol·L -1)和CdCl2(500 mmol·L -1)胁迫条件下表达量的变化。【结果】从甘蔗品种ROC22中克隆获得一个WRKY转录因子基因,命名为ScWRKY6(GenBank登录号为MH393927)。该基因序列全长1 289 bp,包含1个1 059 bp的ORF,编码352个氨基酸,并具有45个磷酸化位点;理论等电点为9.73,不稳定指数为50.23,亲水性为-0.579,推测其为碱性不稳定亲水性蛋白。ScWRKY6蛋白具有1个WRKY结构域和1个锌指结构域(CX5CX23HXH),该蛋白氨基酸序列与高粱(Sorghum bicolor)WRKY(XP_002464211.1)的同源性最高,根据系统进化树分析可以推测其属于WRKY家族Ⅱd亚类。亚细胞定位结果显示,ScWRKY6::GFP融合蛋白定位在细胞核上。酵母转录激活验证试验显示,ScWRKY6蛋白不具有转录自激活活性。qRT-PCR分析表明,ScWRKY6在甘蔗中为组成型表达,其表达量由大到小依次为蔗芽、叶、根、皮和蔗髓,其中蔗芽、叶和根的表达量分别为蔗髓的2.05、1.55和1.37倍。ScWRKY6在NaCl、PEG、MeJA、重金属Cu 2+和Cd 2+胁迫诱导下表达量均上调,其在NaCl处理12 h时表达量最高,为对照的4.18倍;在PEG处理3 h时表达量最高,为对照组的6.88倍;在CuCl2和CdCl2诱导24 h时表达量最高,分别为对照组的3.63倍和4.86倍。【结论】ScWRKY6蛋白定位在细胞核上,不具有转录自激活活性;该基因在甘蔗不同组织部位中均有表达,且受NaCl、PEG、CuCl2和CdCl2等胁迫的诱导,推测ScWRKY6可能在甘蔗干旱应答、耐盐及金属离子胁迫响应中发挥作用。
张旭,凌辉,刘峰,黄宁,王玲,毛花英,李聪娜,汤翰臣,苏炜华,苏亚春,阙友雄. 一个甘蔗Ⅱd类WRKY转录因子基因的克隆和表达分析[J]. 中国农业科学, 2018, 51(23): 4409-4423.
ZHANG Xu,LING Hui,LIU Feng,HUANG Ning,WANG Ling,MAO HuaYing,LI CongNa,TANG HanChen,SU WeiHua,SU YaChun,QUE YouXiong. Cloning and Expression Analysis of a Ⅱd Sub-Group WRKY Transcription Factor Gene from Sugarcane[J]. Scientia Agricultura Sinica, 2018, 51(23): 4409-4423.
表1
引物序列及用途"
引物名称Primer name | 引物序列Primer sequence (5°-3°) | 用途Usage |
---|---|---|
ScWRKY6 | F: GCTGCTGAGGGAGTTGCATA | 扩增全长 Full length amplification |
R: AAGCAGGGCCAAACATCTCA | ||
GFP-ScWRKY6 | F: GGGGTACCATGGAGGAAGTGGAGGAGGC | 亚细胞定位 Subcellular localization |
R: GCTCTAGACACCTGTGCTGACTGAGTTGGC | ||
BD-ScWRKY6 | F: CATGCCATGGAGATGGAGGAAGTGGAGGAGGC | 转录激活 Transactivation |
R: CGCGGATCCCACCTGTGCTGACTGAGTTGGC | ||
qScWRKY6 | F: GCGAGGTCCTTCTTGTCGTC | 实时荧光定量PCR qRT-PCR |
R: CGTTCACCGGATCGCTCATT | ||
CAC | F: ACAACGTCAGGCAAAGCAAA | 内参基因 Actin gene |
R: AGATCAACTCCACCTCTGCG | ||
CUL | F: TGCTGAATGTGTTGAGCAGC | 内参基因 Actin gene |
R: TTGTCGCGCTCCAAGTAGTC |
图4
ScWRKY6与其他植物WRKY氨基酸序列比对 玉米:Zea mays,ACG25409.1;高粱:Sorghum bicolor,XP_002464211.1;黍:Panicum miliaceum,PAN46075.1;狗尾草:Setaria viridis,XP_004982383.1;二穗短柄草:Brachypodium distachyum,XP_010232453.1;粗山羊草:Aegilops tauschii,XP_020149328.1;大麦:Hordeum vulgare,BAJ85995.1;小麦:Triticum aestivum,ACD80360.1;水稻:Oryza sativa,BAT17937.1"
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