黑果枸杞WD40编码基因LrAN11的克隆及表达分析

Cloning and Expression Analysis of LrAN11 Encoding a WD40 Protein in Lycium ruthenicum

WD40蛋白广泛存在于真核生物中,是一类高度保守的蛋白家族,具有广泛的生物化学和细胞生物学功能。为探索黑果枸杞LrAN11的功能,采用同源克隆的方法克隆了黑果枸杞WD40蛋白基因,将其命名为LrAN11,Gen Bank登录号:KY131959。生物信息学分析结果表明,该基因的编码区长1 029bp,编码342个氨基酸,蛋白分子量为38.3 kD,理论等电点为4.95,含有5个WD40基序,属于WD40类蛋白基因家族;经预测LrAN11定位于细胞质中,该基因编码的氨基酸序列具有较高的保守性,与马铃薯、番茄、甜椒和美花烟草具有较近的亲缘关系;定量RT-PCR检测表明,LrAN11基因在黑果枸杞各组织中均有表达,其中在青果中的相对表达量最高,在根和紫果中次之,而在黑果中表达最低,表明LrAN11可能参与黑果枸杞花青素的生物合成的调控;在韩国枸杞和0901枸杞品种中表达显著,在其他14个枸杞品种中表达均较低且无显著差异性(P>0.05),说明LrAN11的表达具有品种特异性。此外,随着NaCl处理时间的延长,该基因的表达量先降低后升高,且在2、12和24 h的表达量与对照相比存在显著差异性(P<0.05),说明LrAN11可能参与黑果枸杞对盐胁迫的响应。本研究结果为进一步阐明黑果枸杞LrAN11基因的功能及其在黑果枸杞遗传改良中的应用奠定了基础。

WD40 protein, also known as WD repeat protein, is widely found in eukaryotes and is a highly conserved protem family. To explore the function of LrANI 1 in Lycium ruthenicum, the fruit of Lycium ruthenicum was used as experimental material. The WD40 gene of L. ruthenicum was cloned by homology cloning and PCR, and named as LrANI 1. Its GenBank accession number was KY131959. The results of bioinformatics analysis showed that the coding region of this gene was 1 029 bp, encoding 342 amino acids. The proten has a weight of 38.3 kD and the isoelectric point is 4.95. It contains five WD40 motifs, belongs to WD40 family. It is predicted that LrAN11 encoding protein is located in the cytoplasm. Quantitative RT-PCR showed that LrAN11 gene was expressed in roots, stems, leaves, flowers and fiuit （fruit, purple fruit and black fruit） , and the relative expression of LrANll in green fruit was the highest, the relative expression in root and purple fruit was the second, while it was almost absent in L. Ruthenicum, which indicated that LrAN11 may be involved in the regulation of anthocyanin biosynthesis in L. ruthenicum. LrAN11 has high expression level in South Koreag wolf＇oerry and 0901 , but low in the other 14 varieties of wolfberry （ P 〉 0.05 ） , indicate that the expression of LrAN11 has a variety specificity. With the treatment time of NaCl, the expression level of LrANI 1 was decreased at first and then increased, and there was significant difference（P 〈 0.05） after 2, 12 and 24 hours treatment when compared with control, indicated that LrAN11 may be involved in the response of salt stress in L. ruthenicum. This study laid a foundation for further elucidating the function of LrAN11 gene of L. ruthenicum and its application in the genetic improvement of L. ruthenicum.