Cloning And Functional Analysis Of GhFBP And GhVIN2Genes Related To Fiber Development In Cotton(Gossypium Hirsutum L.)

Cotton (Gossypium spp) is one of the most important economic crops worldwide. Fiber is from a single cotton ovular epidermal cell. Due to excellent properties, more and more people make use of it, nowadays the researches on fiber are increasing. However, cotton fiber development is a very complex process with thousands of genes participated in. Cotton fiber developmental mutants are the elite material for elucidating the molecular mechanism of fiber developmen. By comparing mutant type with wild type in fiber developmental process, key genes responsible for fiber development can be further confirmed.This research mainly focused on two genes:GhFBP and GhVIN2, which both had a significant expression difference between G. hirsutum acc. TM-1and im mutant by hybridization chip analysis. Further, we cloned these two genes, and analyzed their structures and preliminary function. The results are as following:1. Cloning and functional analysis of GhFBPIn plant, GhFBP is an important enzyme in biosynthesis pathway. It catalyses fructose-1,6-biphosphate into6-frucphosphate and Pi, which6-frucphosphate is a necessary hexose for sucrose synthesis. In this paper, we choose the gene’s EST sequence with significant expression difference from hybridization chip, search the homologous sequence on the internet, then clone this gene with RT-PCR. As a result, we got the cDNA sequence and genomic DNA sequence of this gene. The cDNA sequence includes a complete open reading frame (ORF) with1026bp length, encodes341amino acids. Its PI is5.86, and Mw is37.15KD.This gene contains12extrons and11introns. Chromosome location showed that one copy of the gene was located on D2chromosome in tetraploid cotton species by SNAP marker analysis. Expression analysis showed that GhFBP predominantly expressed in cotton fiber, especially after16DPA, it’s expressional level increases fast. In 16DPA-19DPA, the gene’s expressional level in TM-1is higher than that in im mutant, implying their association with fiber quality difference between TM-1and im. Further, sucrose content were detected the significant difference between TM-1and im, which is also related the expression difference of GhFBP in the two materials.2. Cloning and functional analysis of GhVIN2In plant vacuole, GhVIN2can catalyze sucrose into glucose and fructose, which affect the cell’s osmotic pressure, accelerate fiber cells’elongation. In this paper, we choose the gene’s EST sequence with significant expression difference from hybridization chip, search the homologous sequences on the internet, and then find a homologous gene submitted in NCBI. Further, we design special primers for the gene and clone it with RT-PCR. As a result, we got the cDNA sequence and genomic DNA sequence of this gene, The cDNA sequence includes a whole ORF with1587bp length. It encodes618amino acids. PI is5.25, Mw is69.KD. Chromosome location showed that one copy of the gene was located on A3chromosome in tetraploid cotton species by SNAP marker analysis. Expression analysis showed that GhVIN2predominantly expressed in cotton anther, in cotton fiber, the transcripts of GhVIN2are highest at13DPA. After13DPA, it’s expressional level dropped fast.During13DPA-19DPA, the gene’s expressional level in TM-1is higher than that in im mutant. Using CSIL028×im F2/F2:3populations, association analysis showed that GhVIN2was responsible for one fiber trait:fiber strength. The result indicated that GhVIN2plays an important role in fiber elongation and secondary wall thickening in the process of fiber development.