The Preliminary Analysis Of Invertase Gene Regulating The Heat Tolerance Of Tomato Fruit

Invertase (INV, EC3.2.1.26) catalyzes the irreversible hydrolysis of sucrose to glucose and fructose in higher plants, thereby playing key roles in plant suger metabolism. Tomato (Solarium lycopersicum Mill.) is one of most widely cultivated vegetables in the world, and a kind of classical model plant to research on fruit development and ripening process as well. Tomato is thermophilic, but can’t tolerate high temperature. In recent years, heat stress has become an important factor limiting tomato production in summer. Tomato come about flower dropping, fruit dropping or ovary abortion when exposured to heat stress. This may be caused by that photoassimilate transporting to flower bud and young fruit impeded, or the capability of sucrose utilization declined. What plays a key role in the assimilate transportation and utilization is sucrose metabolism, especially the process of sucrose into hexose (glucose and fructose). Consequently, invertase might be the important factor to regulate the heat resistance of tomato fruit.The previous research came from our group showed that cell wall invertase (CWIN) and vacuolar invertase (VIN) played key roles in the heat resistance of tomato fruit. Besides, the Lin7gene coding for tomato CWIN is closely related to the heat resistance of tomato young fruit. Meanwhile relevant experiments showed unknown new genes coding for tomato VIN may still undiscovered, and they may participate in the regulation to heat resistance in tomato fruit. Based on these, the research contents and results of this paper are listed below: 1. Cloning of Lin9and expression analysis of Lin9from tomato young fruit after heat stressA potential tomato invertase gene (Lin9) were searched out from NCBI database. Both the cDNA and genomic DNA of Lin9were cloned and sequenced. The gDNA was4012bp in length which harbored seven exons and six introns. It contained1959bp open reading frame which encoded652putative amino acids. The putative Lin9protein belonged to stable hydrophilic protein. The invertase characteristic functional domain named glycoside hydrolase family32exist in the Lin9protein. Sequence analysis showed Lin9belonged to vacuolar invertase gene. Real-time fluorescence quantitative PCR revealed that Lin9was expressed highest in the flower, while Lin9transcripts were all in the extremely low expression level in the other tissues. Compared with the known tomato vacuolar invertase gene TIV1, Lin9transcripts had a low expression level as a whole. This result indicated that Lin9may mainly play an role in the development of floral organ. A heat-tolerant (HT) and a heat-sensitive (HS) tomato line were used to analysis the expression pattern of Lin9from tomato young fruit after heat stress by the RT-PCR and qRT-PCR. The increased Lin9expression abundance in two lines revealed that Lin9responded to heat stress in tomato young fruit. Besides, the increased Lin9expression abundance in HT was markedly higher than that in HS. It was speculated that Lin9may further participate in the regulation to heat resistance in tomato young fruit.2. Cloning of Lin7gene, heterogeneous expression analysis of promoter and transgenic tomato plants were acquiredGene-specific primers were designed for PCR amplification, then HT Linl gene promoter, Lin7gene coding sequence (CDS) from HT and HS were amplified. Sequence analysis showed the Lin7gene coding sequence (CDS) from HT and HS was both1752bp in length which encoded583putative amino acids. Sequence alignment showed, compared with the HS, there were two single-bases mutation exist in HT Linl CDS. One of the two mutations (Gâ†'A) resulted in amino acid change of Glu (E)â†'Lys (K) at553rd. The difference between HT Lin7-CDS and HS might result in relevant differences of gene function. The HT-Lin7promoter and GUS gene were fused for the construction of expression vector. The fused genes were transformed to Arabidopsis thaliana. The results of GUS staining show that the Lin7promoter owned a high expressing activity in anther and stigma of Arabidopsis thaliana. Taken pBI121as original expression vector, we replaced original CaMV35S promoter and GUS gene with HT Lin7native promoter and HT Lin7gene, respectively. The recombinant vector pBI-HT-Lin7which HT Lin7native promoter drive Lin7gene expression was constructed. Based on previous experimental method, an efficient tomato regeneration and genetic transformation system was established by exploring the suitable condition of isolated culture for HS tomato cotyledons. The expression vector pBI-HT-Lin7contained target gene HT-Lin7was transferred into HS tomato genome through Agrobacterium tumefaciens-mediated leaf-disc transformation method. Eleven To transformation lines were generated finally. Through PCR analysis using gene-specific primers for NPTâ…¡,10lines among them were identified as transgenic positive plants. At present the10T0transgenic lines are growing normally in a greenhouse, and part seeds of them have be harvested.In conclusion, this study cloned a new gene Lin9for the first time. The expression analysis result showed Lin9was closely related to heat tolerance of tomato young fruit. On the other hand, this study expressed the HT Lin7gene in the HS tomato by transgenic method to test if this would confer heat tolerance in the HS line. The To transgenic lines have be generated and part seeds of them have be harvested. This provided a solid foundation for obtaining the direct evidence that Lin7gene improving heat tolerance of tomato fruit for the next stage. The ultimate goal of these researches is to illuminate the molecular mechanism how invertase regulate the heat tolerance of tomato fruit after heat stress.