番茄Trihelix家族SIP1亚家族基因(SlGT-33)克隆表达分析及功能研究

Cloning expression analysis and functions of SIP1 subfamily gene(SlGT-33)in tomato Trihelix family

【目的】对番茄三螺旋(Trihelix)家族SIP1亚家族基因(SlGT-33)进行克隆表达及功能研究,为深入解析SIP1亚家族成员调控植物生长发育机制及选育矮化番茄品种提供理论依据。【方法】以番茄品种AC++为材料,PCR扩增SlGT-33基因,对其进行序列分析,采用实时荧光定量PCR(qRT-PCR)检测SlGT-33基因在不同组织及外源激素和非生物胁迫下的表达模式,并利用RNAi技术鉴定SlGT-33基因的生物学功能。【结果】扩增获得的SlGT-33基因(GenBank登录号Solyc12g043090)开放阅读框(ORF)为1125 bp,编码374个氨基酸残基,与已知同源基因序列(GenBank登录号XP004252336.1)仅缺少3个碱基。SlGT-33与SlGT-17(GenBank登录号Solyc05g018350)位于同一分支,虽然二者的氨基酸序列相似性最高,但仅为45.8%。SlGT-33基因在茎和成熟叶中的相对表达量较高,显著高于其他组织(P<0.05,下同);经IAA、GA3和MeJA处理后,SlGT-33基因的相对表达量均与对照无显著差异(P>0.05,下同);SlGT-33基因经ABA处理2~24 h时相对表达量均显著高于对照。SlGT-33基因在高盐胁迫和机械损伤下的相对表达量与对照无显著差异;高温胁迫和低温胁迫下SlGT-33基因表达整体上均呈逐渐降低的变化趋势;在脱水胁迫下SlGT-33基因表达整体上均呈逐渐升高的变化趋势。通过农杆菌介导转化法获得6个SlGT-33-RNAi沉默株系,沉默效率为64%~83%。生长70 d的SlGT-33-RNAi沉默株系RNAi-4和RNAi-5幼苗株高和节间长度均为野生型的60%左右,且复叶结构尺寸明显变小,花序提前形成。茎尖组织中顶端分生组织关键转录因子基因KNOX2和WUS基因在SlGT-33-RNAi沉默株系的相对表达量均极显著(P<0.01)或显著高于野生型,腺苷酸异戊烯转移酶(CTK合成的关键酶)编码基因IPT2和茎尖生长点调控基因PHAN基因在2个SlGT-33-RNAi沉默株系的相对表达量均显著低于野生型。顶端分生组织关键转录因子基因KNOX1基因在2个SlGT-33-RNAi沉默株系的相对表达量与野生型无显著差异。SlGT-33-RNAi沉默株系茎尖组织的细胞分裂素(CTK)含量显著低于野生型。【结论】SlGT-33基因属于环境敏感型基因,其表达受ABA和脱水胁迫诱导,但受极端温度的抑制。抑制SlGT-33基因表达会导致了番茄植株矮化和生殖生长加速,其作用机制与顶端分生组织的CTK合成受抑制及茎尖组织调控基因KNOX2、PHAN和WUS的异常表达密切相关。

【Objective】Cloning expression and function of SIP1 subfamily gene(SlGT-33)of tomato Trihelix family to provide theoretical basis for analyzing the mechanism of SIP1 subfamily members regulating plant growth and development,and breeding of dwarf tomato varieties.【Method】Using the tomato variety AC++,amplified its SlGT-33 gene by PCR,performed sequence analysis,and used real-time fluorescence quantitative PCR(qRT-PCR)to detect its expression patterns in different tissues and exogenous hormones and non-biological stress.Finally,the RNAi technique was used to identify the biological function of SlGT-33 gene.【Result】The length of SlGT-33(GenBank accession number:Solyc12 g043090)open reading frame(ORF)was 1125 bp,encoding 375 amino acids residues,which was short of 3 bp than its homologous gene(GenBank accession number:XP004252336.1).Phylogenetic analysis suggested that SlGT-33 protein was in the same branch with SlGT-17(GenBank accession number:Solyc05 g018350),though the similarity between their amino acids sequence was the highest,the value was only 45.8%.SlGT-33 gene was significantly expressed in stem and mature leaves than other tissues(P<0.05,the same below).After IAA,GA3,MeJA treatments,the expression of SlGT-33 gene did not differ significantly from control(P>0.05,the same below).Compared to control,SlGT-33 could be significantly up-regulated by ABA within 2-24 h.SlGT-33 gene expression was not significantly different from control under high salt stress and mechanical damage.The expression of SlGT-33 gene decreased gradually under high temperature stress and low temperature stress,while expression of SlGT-33 gene increased gradually under dehydrationstress.Six SlGT-33-RNAi silent lines were harvested by Agrobacterium-mediated transformation and silence rate varied from 64%to 83%.The plant height and internode length of 70 d SlGT-33-RNAi silent lines decreased to 60%of wild types.The compound leaf structure was significantly smaller and the inflorescence formed in advance.The key transcription factor genes KNOX2 and WUS in stem tip tissue were extremely significantly expressed in the SlGT-33-RNAi silent lines(P<0.01)or significantly higher than the wild type.Adenylate isopentenyl transferase(key enzyme for CTK synthesis)encoded gene IPT2 and the stem tip growth point regulatory gene PHAN were expressed significantly lower in two SlGT-33-RNAi silent linesthan wild type.The expression of the apical biological tissue key transcription factor gene KNOX1 gene was not significantly different compared with in two SlGT-33-RNAi silentlines.Thecytokinin(CTK)of SlGT-33-RNAi silent line stem tip tissue was significantly lower than in the wildtype.【Conclusion】SlGT-33 gene was sensitive to environment.It can be induced by ABA and dehydration stresses and suppressed by extreme temperature.But suppressed expression of SlGT-33 leads to dwarf plants and accelerating reproductive development,which is related with inhibition of CTK synthesis of apical meristem and abnormal expressions of stem tip tissue key regulation genes KNOX2,PHAN and WUS.