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茶树亚硝酸还原酶基因CsNiR的克隆及表达分析

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以‘龙井43’茶树叶片的cDNA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 kD,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,CsNiR具有完整的NiR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-1NH4NO3),qRT-PCR测定发现,正常氮素处理的2h和6h,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CsNiR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.
Molecular Cloning and Expression Analysis of Nitrite Reductase Gene CsNiR in Tea Plant
Teanitrite reductase (CsNiR) was cloned by RT-PCR from cDNA 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 NiR 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 kD and theoretical pI 6.12.Prediction results by InterProScan 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.qRT-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 qRT-PCR after resupplying normal N (1 mmol · L-1 NH4NO3) and low N (0.1 mmol · L-1 NH4NO3) 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.

Camellia sinensisnitrite reductasecloninggene expressionnitrogen

张芬、王丽鸳、成浩、韦康、胡娟、张成才、刘圆、吴立赟、李海琳

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中国农业科学院茶叶研究所,国家茶树改良中心,杭州310008

茶树 亚硝酸还原酶 克隆 基因表达 氮素

国家自然科学基金国家现代农业产业技术体系建设专项资金项目浙江省农业新品种选育重点专项

31570695CARS-232012C2905-4

2016

园艺学报
中国园艺学会 中国农业科学院蔬菜花卉研究所

园艺学报

CSTPCDCSCD北大核心
影响因子:1.127
ISSN:0513-353X
年,卷(期):2016.43(7)
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