茶树亚硝酸还原酶基因CsNiR的克隆及表达分析  

Molecular Cloning and Expression Analysis of Nitrite Reductase Gene CsNiR in Tea Plant

作　　者：张芬[1] 王丽鸳[1] 成浩[1] 韦康[1] 胡娟[1] 张成才[1] 刘圆[1] 吴立赟[1] 李海琳[1] 

机构地区：[1]中国农业科学院茶叶研究所,国家茶树改良中心,杭州310008

出　　处：《园艺学报》2016年第7期1348-1356,共9页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(31570695);国家现代农业产业技术体系建设专项资金项目(CARS-23);浙江省农业新品种选育重点专项(2012C2905-4)

摘　　要：以‘龙井43’茶树叶片的c DNA为模板,利用RT PCR技术克隆了茶树亚硝酸还原酶基因(CsNiR),得到完整的ORF全长为1 764 bp,编码587个氨基酸;所推导的氨基酸序列与垂枝桦(Betula pendula)、拟南芥(Arabidopsis thaliana)、菠菜(Spinacia oleracea)和水稻(Oryza sativa)的同源性均大于76%。生物信息学分析表明,CsNiR分子量为68.648 k D,等电点为6.12,为亲水性的非分泌蛋白;二级结构的预测显示,Cs Ni R具有完整的Ni R蛋白结构,含血红素蛋白β–化合物区域和4Fe-4S区域。实时荧光定量结果表明,该基因在成熟叶片中表达量高于一芽二叶和根。采用营养液水培3个茶树品种,在氮饥饿两周后分别供应正常氮素和低氮素(1和0.1 mmol·L^(-1) NH4NO3),q RT-PCR测定发现,正常氮素处理的2 h和6 h,诱导根CsNiR表达量显著增加,叶片中的表达量变化延迟到24 h之后,且变化幅度存在基因型之间的差异;低氮处理对CsNiR表达量的影响相对较小。因此,研究Cs Ni R基因的表达特性及其在茶树氮素利用中所发挥的作用,需结合茶树基因型、组织部位、供氮水平等进行综合评价。Teanitrite reductase（CsNiR）was cloned by RT-PCR from c DNA isolated from leaves of cultivar ＇Longjing 43＇.The complete ORF of the CsNiR was 1 764 bpencoding 587 amino acids.Alignment of amino acid sequences showed that CsNiR sharing more than 76% similarities to Ni R in Betula pendula,Arabidopsis thaliana,Spinacia oleracea and Oryza sativa.Bioinformatics analysis indicated that the CsNiR was a hydrophilic non-secretory protein with molecular weight 68.648 k D and theoretical p I 6.12.Prediction results by Inter Pro Scan showed the secondary structure of CsNiR protein comprised of a hemoprotein beta component and a 4Fe-4S region,and its 3D structure was also predicted by Swiss Model.q RT-PCR analysis revealed that the expression abundance of CsNiR in mature leaves was the highest among the three tested tissues（two leaves and a bud,mature leaves and roots）.Meanwhile,the transcript changes of CsNiR responding to different nitrogen（N）levels were studied by q RT-PCR after resupplying normal N（1 mmol·L^-1 NH_4NO_3）and low N（0.1 mmol·L^-1 NH_4NO_3）on hydroponic seedlings of three tea varieties with treatment of two week N starvation.The CsNiR expression levels were increased significantly in roots at 2 h and 6 h under normal N treatment,but they were changed since 24 h after N supplied in leaves.Furthermore,the transcription of CsNiR was also different among varieties.The transcript abundance of CsNiR increased more greatly under the normal N condition compared to those under low N treatment.Thus,factors like genotypes,tissues,nitrogen levels should be taken into consideration for the role of CsNiR in nitrogen utilization in tea plants.

关 键 词：茶树 亚硝酸还原酶 克隆 基因表达 氮素 

分 类 号：S571.1[农业科学—茶叶生产加工]