Cloning, Structure, Phylogeny And Expression Of The Gene Families Involved In Sucrose Metabolism In Hevea Brasiliensis

Sucrose is the major product of photosynthesis, and also the main form in plants for carbohydrate accumulation, transportation and distribution. In rubber tree, sucrose is synthesised in leaves, and translocated through phloem from photosynthetic leaves (source) into non-photosynthetic tissues (sink), including the rubber-producing laticifers. For its usage in rubber biosynthesis, sucrose in latex (cytoplasm of the laticifers) needs to be degraded into hexoses first. In plants, invertase (INV), sucrose synthase (Sus), sucrose phosphate synthase (SPS) and sucrose phosphatase (SPP) are konown as the enzymes in sucrose metabolism. However, little is known about these gene families in rubber tree. In this study, utilizing the second generation sequencing data of genome and transcriptome, we extensively cloned the isoforms of HbINV, HbSus, HbSPS and HbSPP gene families and analyzed their structure, phylogeny and gene expression patterns. The main results are as follows:1. A total of 17 HbINV genes,6 HbSus genes,4 HbSPS genes and 3 HbSPP genes were cloned, and some of the family members within the families were found to have high sequence similarity.2. In each gene family, the gene structure of family members has high similarity for each other, including the relative position of exons and introns, the length and number of exons. Phylogenically, the families of HbINV, HbSus and HbSPS were clustered into distinct clades, such as the clustering of HbINV genes into ClassN-?, ClassN-?, ClassC and Class?, HbSus genesinto Sus ?, Sus ? and Sus?, and HbSPS genes into Class ?, Class ? and Class?. However, HbSPP genes only have one main clade. The gene members in these clades can be further clustered into monocot and dicot clades, suggesting their evolutionary divergence before the common ancestor of dicots and monocots.3. We analyzed the gene expression of all members of the four gene families in seven tissues (latex, bark, leaf, root, seed, male flower and female flower) using Solexa sequencing and QPCR. Results showed that different genes had different expression patterns, and even the members in same family had strikingly different expression patterns in same tissue. And each tissue had one predominant family member:HbNIN2, HbCIN1, HbVIN3, HbSus3, HbSPS1 and HbSPP1 being the highest expression genes in latex; HbNIN2, HbCIN2, HbVIN2, HbSus4, HbSPS2 and HbSPP1 being the highest expression genes in bark; HbNIN8, HbCIN2, HbVIN2, HbSusS, HbSPS3 and HbSPP1 being the highest expression genes in leaf; HbNIN5, HbCIN2, HbVIN3, HbSus5, HbSPS2 and HbSPP1 being the highest expression genes in root. Gene expression profile also indictaed that the expression levels of the four families were different to each other in different tissues. Overall, the HbSus family had a relatively higher expression level, HbINV and HbSPS took the second place, and HbSPP had a relatively lower level.4. We analyzed the gene expression of all members of the four gene families at different developmental stages of leaf using Solexa sequencing and QPCR. With the development of leaf, some genes in the four gene families displayed distinct changes at expression levels, including the Inv genes of HbNINl, HbNIN2, HbNIN6, HbNIN8, HbCIN3, HbVINl, HbVIN2 and HbVIN3, the Sus genes of HbSus2 to HbSus5, the SPS genes of HbSPS1 to HbSPS4 and HbSPP1. These genes may be related to the sucrose metabolism during leaf development. In addition, the expressions of HbCIN2, HbSus5 and HbSPS3 showed apparent diurnal variation, the expressions of HbCIN2, HbVIN2, HbSus5, HbSPS3 and HbSPP1 were differentially expressed in different types of veins, suggesting their roles in phloem loading in leaf.5. Ethrel can markedly improve the yield of latex, and we here examined the expressions of the four gene families under Ethrel treatment. HbNIN3 and HbVIN3 were up-regulated, but HbSus2 and HbSus3 were down-regulated. In addition, HbNIN3 and HbSus3 were up-regulated by wounding. Further, most genes of the four gene families were up-regulated by tapping, suggesting their roles in latex regeneration.6. We analyzed the expressions of the major members of the four gene families in seedling leaf, root and bark responding to the treatments of high temperature, low temperature and drought condition. The results showed that most of these genes were markedly regulated in different tissues under various stresses, suggesting their involvement in the tolerance to environmental stresses.7. We carried out SSR and SNP analysis on 30 sucrose metabolism-related genes. Among these genes,61 SSRs were identified, including 46 single nucleotide repeat,10 two nucleotide repeat,1 three nucleotide repeat and 4 four nucleotide repeat. Except one SSR was found in the first exon of HbNIN6 the others resided in introns. SNP analysis revealed 3 SNPs in clone PR107,5 SNPs in clone RRIM600,45 SNPs in clone WenChang11(16 SNPs in HbSus6),3 SNPs in clone YunYan774, and no SNPs in RY8-79.The results in this study will help us understand the molecular mechanisms of sucrose metabolism in Hevea, and provide important molecular marker for rubber breeding. Consequently, this work has important theoretical significance and application prospects.