The Effect Of Down-regulating 3-ketosphinganine Reductase Gene(GhKDSR2-1)on The Development Of Cotton Fibers

Cotton is an important economic crop in our country. It has important roles in the domistic economy. It is an important source of natural fiber. Cotton fiber is an extremely elongated single cell, which derived from the epidermis of the ovule. Its development process consists of obviously different yet partially overlapping four stages namely the differentiantion and initiation of fiber cell, elongation, secondary wall deposition and maturation. So fiber cell is an ideal material for the study on plant cell development. Furthermore, since cotton fiber yield and quality depend on the fiber development, to illuminate the regulatory mechanism of fiber growth is an important step to imptrove cotton fiber yield and quality. Although research on cotton fiber development in recent years has made some progresses, but the molecular mechanisms regulating cotton fiber development remains unclear.Sphingolipid is a kind of lipid molecules. As an important component widely present in the membrane, sphingolipid and its derivatives are important in many cellular activities such as dynamic regulation and signal transduction. Different molecules of sphingolipids present in different tissues and even different cells. However, the function of sphingolipid on plant growth and development is still unclear. Because the expansion of membrane area will be required to meet fiber rapid elongation, various sphingolipid molecules may increase to support a variety of physiological and biochemical activities.Therefore sphingolipid synthesis may play an important role in fiber growth.In order to explore the role of sphingolipids in cell growth and development of cotton fiber, we cloned the homologue of 3-keto-dihydro-sphingosine reductase gene(Gh KDSR2) from upland cotton. By the transgenic cotton plants down-regualting Gh KDSR2-1 expression level, we analyzed the functions of the Gh KDSR2-1 in cotton plants, especially in the fiber growth and development. The main results are as follows:1. Cloning and sequence analysis of Gh KDSR2 geneTwo homologous Gh KDSR2 sequences were cloned from wildtype cotton Xuzhou-142 10 DPA-c DNA library. Gh KDSR2-1, contains a 996 bp-length integral ORF, and its deduced amino acid sequence has 331 amino acid residues, protein molecular weight is 36.0k D, isoelectric point is 8.065; Gh KDSR2-2, contains a 999 bp-length integral ORF, and its deduced amino acid sequence has 332 amino acid residues, protein molecular weight is 36.2 k D, isoelectric point is 7.436. They have a high homology to other species’ such as cocoa, Arabidopsis and Castor. These results indicated that the cloned Gh KDSR2 were KDSR homologous genes in upland cotton.2. Expression patterns of Gh KDSR2-1 genesTo illuminate the function of Gh KDSR2-1 in cotton growth and development initially, the expression patterns of Gh KDSR2-1 in wild type plant and fiber mutant were detected by real-time quatitative RT-PCR. The results showed that Gh KDSR2-1 was expressed in the test sample, but has significant differences in their expression levels. Its expression peaked in rapid elongation period during fiber development and indicated that Gh KDSR2-1 has role in fiber development. We then examined the gene expression differences in fiber development mutants. The results showed that Gh KDSR2-1 expression compared to the wild-type has significantly decreased in fiber lint-free floc mutant start of fiber and short fiber mutant rapid elongation. This shows Gh KDSR2-1 in initiation and elongation stages of fiber may play an important role.3. Influence of anti-Gh KDSR2-1 in Cotton Plant GrowthWe use Gh KDSR2-1 sequence to construct antisense inhibition of plant expression vectors and transfer it into cotton. The transgenic results shows that: small plant slender, slow growth, difficult to survive; small flowers, petals can not be completely open, and less severe pollen abortion, it is difficult to stabilize the fruit; small fruit, slow growth, and each fruit only 1-2 seeds to mature; Leaf color becomes deep and surface is roughness. Then further chlorophyll and lignin contents were tested and found elevated levels of chlorophyll, lignin content increased and the distribution area increased. These results indicate that transgenic cotton plant growth and development was severely disrupted.4. Down-regulating Gh KDSR2-1 inhibits fiber initiation and elongationWe take the ovule of wild-type and anti Gh KDSR2-1 cotton to cultured for 5d,10 d and 15 d in virto.The result show that Suppressing expression of Gh KDSR2-1 inhibits fiber elongation. Moreover, the mature transgenic cotton fibers become shorter compared to the wild type cotton fibers. The results in analysis of-1DPA and 0DPA ovule in transgenic plant of anti-Gh KDSR2-1 by SEM shows that the starting of transgenic fiber cell is inhibited and significantly the initial amount reduced. These results shows that expression of Gh KDSR2-1 played an important role in fiber initiation and elongation.5. Down-regulating Gh KDSR2-1influence the content of oil and proteinTo clear Antisense inhibition of Gh KDSR2 Cotton sphingolipid synthesis, we detected the content of lipids transgenic cotton seeds and leaves. We found that in transgenic leaves and seeds of their fat content have undergone a significant decline compared to the wild type. To further investigate the genetically modified material, the sphingolipid synthesis route is affected, we detect the expression levels of the sphingolipid synthesis related genes in transgenic and wild-type materials materials, The results show that the expression levels of the sphingolipid synthesis related genes have undergone significant changes in anti-Gh KDSR2-1 materials.