马铃薯低温响应的ScmiR390-5p及其靶基因表达与结构分析

Expression and Structural Analysis of SC MI390-5p and Its Target Genes in Potato Response to Low Temperature

【目的】研究马铃薯富含亮氨酸重复序列(leucine-rich repeat receptor-like protein kinase LRR-RLK)类受体蛋白激酶基因SCLRRK1受miR390(ScmiR390-5p)调控响应非生物胁迫的机理。【方法】通过低温响应马铃薯miRNA测序分析与靶基因预测,发现ScmiR390-5p通过调控1个可能的LRR类受体蛋白激酶基因对低温做出响应;利用实时荧光定量PCR技术(Quantitative Real-time PCR,RT-qPCR)验证ScmiR390-5p和SCLRRK1响应低温胁迫的表达情况;利用RLM-5′RACE确定ScmiR390-5p作用于SCLRRK1的切割位点;利用PCR技术克隆得到SCLRRK1的DNA序列和CDS序列,生物信息学分析SCLRRK1的结构和功能;利用RT-qPCR分析ScmiR390-5p/SCLRRK1在马铃薯各组织中的表达情况及其响应各种非生物胁迫的表达情况。【结果】RT-qPCR结果表明,低温诱导ScmiR390-5p表达,SCLRRK1的表达则受到低温的抑制,ScmiR390-5p和SCLRRK1的表达在低温胁迫下呈负相关性;RLM-5′RACE分析表明,ScmiR390-5p作用于SCLRRK1的切割位点是ATTCCT//CCTGAGTT,马铃薯ScmiR390-5p/SCLRRK1调控模块在低温响应中起作用。克隆结果表明SCLRRK1的CDS序列长度为2 685bp,编码894个氨基酸,DNA序列长度为3 549 bp,含有1个内含子、2个外显子、3′非编码区和5′非编码区,ScmiR390-5p的靶位点位于SCLRRK1 CDS序列内部(960—981 bp,GGAACTATTCCTCCTGAGTTT)。生物信息学显示SCLRRK1是1个富含亮氨酸重复序列(leucine-richrepeat,LRR)的类受体蛋白激酶基因,编码的蛋白属于跨膜分泌蛋白。马铃薯组织表达RT-qPCR分析显示,ScmiR390-5p在叶中的表达量最高,根次之,茎中(地上茎、块茎和匍匐茎)表达量较低;而SCLRRK1的表达情况与ScmiR390-5p相反,其在叶中的表达最低,在茎中表达最高(匍匐茎>块茎>地上茎)。多种非生物胁迫结果显示,ScmiR390-5p和SCLRRK1表达在NaCl胁迫下呈负相关,ScmiR390-5p受到NaCl胁迫诱导表达。与对照相比,ABA和6-BA处理下ScmiR390-5p表达先下降之后呈波浪小幅回调;6-BA处理下SCLRRK1表达持续升高,ABA处理下SCLRRK1表达先上升后下降。GA3、PEG和IAA处理8 h时,ScmiR390-5p的表达达到峰值,GA3、PEG和IAA处理都能诱导SCLRRK1表达,但其变化趋势与ScmiR390-5p相关性不强。【结论】SCLRRK1具备编码LRR类受体蛋白激酶的核酸、氨基酸序列和结构基础,是ScmiR390-5p的靶基因;ScmiR390-5p/SCLRRK1调控模块在马铃薯组织器官中具备明显作用;ScmiR390-5p在转录后水平通过抑制马铃薯SCLRRK1的表达对低温胁迫做出响应,同时,马铃薯ScmiR390-5p/SCLRRK1调控模块在NaCl和6-BA胁迫响应中起作用,但不对ABA、GA3、IAA和PEG胁迫做出响应,ScmiR390-5p和SCLRRK1分别在转录水平受到ABA、GA3、IAA和PEG胁迫信号调控。

The study was carried out to investigate the mechanism of potato leucine-rich repeat receptor-like protein kinase(LRR-RLK)receptor protein kinase SCLRRK1 regulated by miR390(ScmiR390-5 p)in response to abiotic stress.【Method】By sequencing and analyzing potato miRNAs in response to low temperature conditions and predicting target genes,we found that ScmiR390-5 p responded to low temperatures by regulating a potential LRR-like receptor protein kinase gene.The expression levels of ScmiR390-5 p and SCLRRK1 in response to low temperature stress were verified by quantitative real-time PCR(RT-qPCR).The cleavage site of ScLRR390-5 p on SCLRRK1 was determined by using RLM-5’RACE.The DNA sequence and CDS sequence of SCLRRK1 were cloned by PCR and the structure and function of SCLRRK1 were predicted by bioinformatics analysis.The expression levels of ScmiR390-5 p/SCLRRK1 in various tissues of potato and under various abiotic stress were analyzed by RT-qPCR.【Result】The results of RT-qPCR showed that the expression of ScmiR390-5 p was induced by low temperature,while the expression of SCLRRK1 was inhibited.There was a negative correlation between the expression of ScmiR390-5 p and scrrk1 under low temperature stress.The result of RLM-5′RACE indicated that cleavage site of ScmiR390-5 p on SCLRRK1 was ATTCCT//CCTGAGTT,and potato ScmiR390-5 p/SCLRRK1 regulatory module was respond to low temperature.The cloning results indicated that the CDS of SCLRRK1 was 2 685 bp in length,encoding 894 amino acids,and the gene sequence was 3 549 bp containing 1 intron,2 exons,3’non-coding region and 5’non-coding region.ScmiR390-5 p target site was located at SCLRRK1 CDS(960-981 bp,GGAACTATTCCTCCTGAGTTT).Bioinformatics analysis showed that the SCLRRK1 encoded a leucine-richrepeat(LRR)-like receptor protein kinase,belonging transmembrane secreted protein.RT-qPCR analysis of potato tissue expression pattern showed that the expression level of the ScmiR390-5 p was highest in the leaves,followed by roots,and relatively lowers in stem(terrestrial stem,tuber and stolon).Differently,the expression level of SCLRRK1 was the lowest in leaves and the highest in stems.The results under various abiotic stresses showed that the expression of ScmiR390-5 p and SCLRRK1 was negatively correlated,and ScmiR390-5 p was induced by NaCl stress.Compared with the control,the expression of ScmiR390-5 p was decreased and then increased slightly after treatment with ABA and 6-BA.The SCLRRK1 level increased continuously under 6-BA treatment,while the expression of SCLRRK1 increased first and then decreased under ABA treatment.The expression of ScmiR390-5 p reached the peak after treatment with GA3,PEG and IAA for 8 h.The expression of SCLRRK1 was induced by GA3,PEG and IAA,but the change trend was not correlated to ScmiR390-5 p.【Conclusion】SCLRRK1 had the amino acid sequence and structural basis of nucleic acid to encode LRR-like receptor protein kinase,which was the target gene of ScmiR390-5 p;ScmiR390-5 p/SCLRRK1 regulatory module had a significant role in potato tissues;ScmiR390-5 p responded to low temperature stress by inhibiting the expression of potato SCLRRK1 at the post-transcriptional level,while ScmiR390-5 p/SCLRRK1 regulated module played a role in salt and 6-BA stress response.The ScmiR390-5 p/SCLRRK1 regulatory module did not respond to ABA,GA3,IAA and PEG stress,and the ScmiR390-5 p and SCLRRK1 were regulated by the above signals at the transcriptional level,respectively.