Cloning And Functional Analysis Of Phosphatidylinositol Transfer Protein (PITP) Homologous Genes In Cotton (Gossypium Hirsutum L.)

Cotton is one of the most important fiber crops in the world.Both yield and fiber quality are the main targets in cotton breeding.Rapid progress in Genetic engineering has provided a new method to improve fiber quality and yield in cotton,and meanwhile set a higher request for the study of the molecular mechanism in fiber growth and in formation of yield and quality.Phosphatidylinositol transfer protein（PITP） is a kind of apolipoproteins which binds phosphatidylmositol（PtdIns） or phosphatidylcholine（PC）.By accelerating transport of PtdIn in cell endomembrane system,PITP is able to modulate site-directed synthesis of several phosphoinositides （PIs）.Therefore,it may be involved in regulation of variously physiological processes and signal pathways,such as lipid transport and metabolism,formation and transportation of secretory vesicle, phospholipase C activity,exocytosis and polar growth.Previous research indicated that PITP homologous gene in cotton is predominantly expressed in fiber.To advance our understanding of the function of PITP in fiber development,four cotton PITP homologous genes were cloned from developing fibers（Ghsh2-1～4）.On the basis of sequence and expression analysis,the relationship between cotton PITP and fiber development was investigated by up- and down- regulation of Ghsh2-1 in transgenic cottons using over-expression and RNA interfering techniques.The main results were as followings:1.Cloning of cotton Ghsh2 homologous genesUsing 3’-RACE（rapid amplification of cDNA ends） method,four cDNA sequences of cotton PITP homologous genes have amplified by querying EST sequences.These cDNA sequences,named as Ghsh2-1～4,contained full-length ORFs of 744bp,747bp,732bp and 783bp,encoding polypetides of 247 aa,248 aa,243 aa and 260 aa,respectively.The genomic sequences of Ghsh2-2 and Ghsh2-4 compassing the ORFs were amplified.The genomic sequences of Ghsh2-2 and Ghsh2-4（1049bp and 3251bp,respectively） both include 6 exons and 5 introns.Except for the first exon,the exon lengths in Ghsh2-2 and Ghsh2-4 were equal,while the introns varied dramatically in length.Multiple sequence alignment indicated that the deduced Ghsh2-1～4 proteins shared high sequence identity with PITPs from yeast and other plants（with identity of 21.1%to 64.0%）.It is also indicated that conserved amino acid residues were distributed among the full-length sequence. The conserved residues included those associated with PtdIns transfer activity（R65,E207,T236 and K239 in Sec14p）,the hydrophobic amino acids involved in formation of Sec14-like hydrophobic pocket,"GlyGly" stablizing the hydrophobic pocket and "YPE" related to Ptdlns binding.In a phylogentic tree of Ghsh2 proteins and other PITP from soybean and Arabidposis, Ghsh2-1～4 were clustered with Ssh2 and Atsec14-19,which may form a new PITP group in plant.2.Expression analysis of Ghsh2 homologous genesQuantitative RT-PCR was employed to detect the expression levels of Ghsh2-1～4 in various organs and tissues.Expression level of Ghsh2-1 and Ghsh2-3 in all investigated tissues is higher than that of Ghsh2-2 and Ghsh2-4.Four Ghsh2 genes were predominantly expressed in later phase of fiber elongation,while Ghsh2-1 showed most specific expression profile.3.Obtaining of cotton transgenic plantsOver-expression vector（p5-Ghsh2-1） and RNA interfering vector（p5-Ghsh2-1RNAi） were transformed into cotton via Agrobacterium-mediated method.Regenerated transgenic cotton plants confirmed by GUS staining included 6 independent p5-Ghsh2-1 transformants（Ghsh-1～6） and 4 p5-Ghsh2-1RNAi transformants（RNAi-1～4）.Quantitative RT-PCR analyses demobstrated that the Ghsh2-1 expression level was significantly down-regulated in RNA interfering transgenic plant（RNAi-2）,while up-regulated in overexpression transformants（Ghsh-3 and Ghsh-6）.4.Effects of altered Ghsh2-1 expression on fiber traitsBy comparing fiber trait of transgenic plants and null segregants in T₁ generation,we found no significant variation in fiber down-regulating Ghsh2-1,while the transgenic plants overexpressing Ghsh2-1（Ghsh-3 and Ghsh-6） showed obvious improvement in fiber-length,fiber-strength and fiber-fineness.Microscopic observation indicated that the early-stage fiber initials of Ghsh-6-82 were longer than that of control,suggesting that up-regulation of Ghsh2-1 might promote fiber elongtion.