棉花烟酰胺合成酶基因GbNocotin及其启动子的克隆和功能分析

Cloning and Functional Analysis of a Cotton Nicotinamide Synthetase Gene GbNocotin and Its Promoter

【目的】克隆棉花烟酰胺合成酶基因及其启动子,明确其表达特征,分析其在转基因育种中的应用前景。【方法】根据对一个棉花Maxxa BAC克隆(78L16)的测序结果,首先从海岛棉品种海7124中PCR扩增获得了棉花烟酰胺合成酶基因GbNocotin的启动子序列,并利用网上数据库PLACE对该序列进行调控元件的预测分析。其次构建该启动子与GUS连接的重组载体pGbNocotin::GUS,并通过花浸染法转化拟南芥并获得转基因植株,分别在幼苗期和成熟期对转基因植株进行GUS染色分析。然后通过RT-PCR获得GbNocotin的开放阅读框(ORF)序列,并利用Mega5.0对GbNocotin进行进化树分析,最后利用实时荧光定量PCR(Real Time PCR)对该基因进行组织表达,诱导表达分析。【结果】GbNocotin的启动子区为1.8 kb,通过相似性比较预测发现该启动子含有1个专一性Fe缺乏诱导元件IRO2OS,还含有干旱、重金属、病原物等逆境响应元件以及植物激素响应元件等。对转化pGbNocotin::GUS拟南芥植株的GUS染色结果表明,在幼苗期GUS基因主要在根部以及下胚轴处表达,而在成熟期除了在根部表达外,还在果荚的基部、花序以及叶柄基部表达。GbNocotin的开放阅读框含有864个核苷酸,编码287个氨基酸,该蛋白等电点为6.76,分子量为32.7 kD。虽然在第11—286位氨基酸处具有NAS保守结构域(PFAM accession number:PF03059),但是与其他植物来源的该类基因相似性较低,与相似度最高的拟南芥的ATNAS3也仅有43%的相似性。GbNocotin的表达具有器官差异性,在根和茎中的表达最强,其次是棉纤维和花,但是在叶片及胚珠中表达量很低。另外,该基因在缺铁条件下表达量显著上升,在缺铁处理一周后棉花幼根中的表达量较对照增加9倍。而CuSO4、PEG、脱落酸(ABA)以及赤霉素(GA)处理均抑制其表达,但是抑制程度不同。尽管4种条件处理24 h后都会使GbNocotin的表达量显著降低,但是CuSO4和ABA的抑制效果最为显著。而48 h后,PEG和GA处理基因的表达量得到恢复,但是CuSO4和ABA处理表达量仍然较低。【结论】GbNocotin的表达具有器官差异性,并受铁缺失诱导以及胁迫条件抑制。该基因与棉花Fe的吸收可能密切相关,有潜在的应用价值。

[Objective] Cloning and characterizing the expression pattern of a cotton nicotinamide synthase gene and its promoter to analyze it＇s application in transgenic crop breeding. [ Method ] According to the sequencing results of a clone （78L 16） of Maxxa BAC, the promoter of a cotton nicotinamide synthase gene GbNocotin was obtained firstly. The online database PLACE was utilized to predict regulatory elements on the promoter. Its recombinant vector pGbNocotin：：GUS was constructed and wastransformed into Arabidopsis by flower dip method and the transgenic plants was obtained, which were used for GUS staining analysis at the seedling and mature stages. Then the open reading frame （ORF） of GbNocotin was obtained by RT-PCR. Phylogenetic tree was made by mega5.0 and expression analysis in different organs and by stress treatments was conducted by real time-PCR. [Result] A specific Fe-deficiency-related element IRO2OS, several stress-responsive such as dehydration, heavy metal and pathogen related elements and phytohormone responsive elements are present in the 1.8Kb promoter region of GbNocotin. GUS staining analysis of transgenic Arabidopsis plants by pGbNocotin：：GUS showed that GUS gene was strongly expressed in the root and hypocotyls at the seedling stage, while it was also detected at the base of pods, inflorescence and petioles as well as root in the flowering Arabidopsis plants. GbNocotin has an ORF with 864 nucleotides and encodes a protein of 287 amino acids, its isoelectric point is 6.76 and the predicted molecular weight is 32.7 kD. Although the conserved NAS domain （PFAM accession number： PF03059） was located at amino acid of 11-286, but the similarity of GbNocotin with homogenous genes from other plant is very low, with the highest similarity of ATNAS3 in Arabidopsis is only 43%. GbNoeotin exhibits an organ differently expressed pattern. It was strongly expressed in root and stem, moderately expressed in cotton fibers and flowers, in contrast to weakly expression levels in leaves and ovule. In addition, Fe deficiency induced the expression of GbNocotin significantly, as the expression level increased as 9 fold compared to the control plants after one week with the treatment of Fe deficiency. Although the treatments of CuSO4, PEG, abscisic acid （ABA） and gibberellin （GA） all depressed the expression of GbNocotin, but the content varied largely, as the expression all decreased dominantly at 24h by the 4 treatments but the inhibitory effects of ABA and CuSO4 were most significantly. And after 48 h, the expression level by PEG and GA treatments has been restored, while that of the CuSO4 and ABA treatments was still very low. [Conclusion] GbNocotin was differentially expressed in various tissues and organs. It was induced by Fe deficiency and depressed by CuSO4, PEG, ABA and GA treatments. The GbNocotin gene may participate in the absorption and utilization of Fe in cotton and may have potential applications in breeding.