水稻氨基酸透性酶基因OsAAP14启动子克隆及时空表达特性分析

Promoter sequence cloning and spatiotemporal expression pattern of rice amino acid permease gene OsAAP14

  • 摘要: 【目的】克隆水稻氨基酸透性酶基因OsAAP14的启动子序列并分析其时空表达特性,为解析氨基酸转运基因生物学功能及了解水稻对有机氮源的响应和氨基酸吸收利用提供理论依据。【方法】PCR扩增OsAAP14基因的启动子序列,并与pCAMBIA1391Z空载体质粒连接构建出启动子-GUS表达载体,并通过农杆菌介导侵染水稻中花11愈伤组织,获得OsAAP14基因的启动子-GUS转基因植株,通过对转基因植株不同组织部位进行GUS染色,并采用石蜡切片技术观察各组织细胞部位表达,经5种不同氨基酸处理后进行根部GUS染色,结合实时荧光定量PCR检测OsAAP14基因在GUS染色部位的相对表达量,最后综合分析该基因的时空表达特性。【结果】克隆获得OsAAP14基因启动子序列为1993 bp,与参考序列日本晴OsAAP14(LOC_Os04g56470)序列一致。该启动子序列含有MBS、P-box、ABRE、CGTCA-motif等激素或胁迫响应元件。从OsAAP14基因的启动子-GUS植株T0代中鉴定获得16个阳性转基因株系。T1代材料不同组织部位GUS染色及实时荧光定量PCR结果均显示,OsAAP14基因在水稻芽伸长的基部和叶片相对表达量较高,在根、叶鞘和穗也有一定表达,但在茎中的相对表达量最低。石蜡切片分析结果显示,OsAAP14基因在根部皮层薄壁细胞、叶片的叶肉细胞、穗的颖壳内部细胞有较高的表达。碱性氨基酸的组氨酸处理下根中的OsAAP14基因表达量随着处理时间的增加显著提高(P<0.05,下同),且赤霉素和脱落酸处理下,根中的OsAAP14基因相对表达量也显著提高。【结论】OsAAP14基因在水稻不同组织均有表达,正常情况(未处理)下在水稻基部和叶片中相对表达量较高,但外源氨基酸和激素处理时,在根中该基因被诱导表达上调,说明OsAAP14基因正常情况下可能主要参与调控水稻地上部分氨基酸的运输,但当外界氨基酸和激素含量增加时则参与调控水稻根部氨基酸的运输。

     

    Abstract: 【Objective】 The objective of this study was to clone the promoter sequence of the rice amino acid permease gene OsAAP14 and analyze its spatiotemporal expression characteristics, which laid a foundation for the analysis of the biological function of the amino acid transport gene and provided a basis for understanding the response of rice to organic nitrogen sources and the absorption and utilization of amino acids.【Method】 The promoter sequence of the amino acid permease gene OsAAP14 was amplified in vitro by PCR technology, the promoter-GUS expression vector was constructed by pCAMBIA1391Z empty carrier plasmid, and the agrobacterium-mediated method was used to transform rice ZH11 callus, and the promoter-GUS transgenic plant of OsAAP14 gene was obtained, and the expression of specific cell sites of each tissue was observed by GUS staining of each tissue site and paraffin sectioning technology. The root GUS staining was car-ried out by five different amino acids treatments, and the gene expression corresponding to the above GUS staining site was further detected by combining real-time fluorescence quantification PCR technology, and the spatiotemporal expression characteristics of the gene were comprehensively analyzed.【Result】The OsAAP14 gene promoter sequence of 1993 bp was obtained by cloning and was consistent with the sequence of the reference sequence Nipponbare OsAAP14 (LOC_Os04g56470). This promoter sequence contained hormone or stress response elements such as MBS, P-box, ABRE and CGTCA-motif. Sixteen positive transgenic lines were identified from the promoter-GUS plants of OsAAP14 gene in T0 generation. GUS staining and real-time fluorescence quantitative PCR results of different tissue parts of T1 generation materials showed that OsAAP14 gene was highly expressed in the basal parts of rice shoot elongation and leaves, and was also expressed in roots, leaf sheaths and panicles, but was least expressed in culms. Paraffin section analysis showed that OsAAP14 gene was highly expressed in root cortex parenchyma cells of roots, mesophyll cells of leaves, and inner cells of glumes of panicles. The expression of OsAAP14 gene in roots under histidine treatment in basic amino acids increased significantly(P<0.05, the same below) with increasing treatment time, and the relative expression of OsAAP14 gene in roots under gibberellin and abscisic acid treatments was also significantly increased.【Conclusion】The amino acid transport gene OsAAP14 expresses in all tissues of riceunder normal environment, and the expression is higher in the basal part and leave of rice. The expression of OsAAP14 in the root is strongly promoted by the exogenous amino acid and hormone treatments, indicating that the OsAAP14 gene may mainly participate in regulating the transportation of amino acids in the aboveground part of rice under normal environment, but it participates in regulating the transportation of rice root amino acidswhen the content of external amino acids and hormones increases.

     

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