| As one of the most important natural fiber crops, cotton plays an important role in the demestic economic of China, since China is the major cotton-producing and consuming country in the world. Cotton fiber is the main product of cotton, and the yield and quality of the mature fiber depends on its growth and development process. Meanwhile, as an extremely elongated plant cell, cotton fiber is an ideal material for the study on plant cell differentiation, growth and secondary wall thickening. So the research of cotton fiber has important theoretical significance and application value. Cotton fiber cell is single-celled and originates from the epidemic of the outer integument of ovule. Fiber cell development includes four discrete but overlapped stages:initiation, elongation, secondary cell wall accumulation and maturation. In recent years, many studies focused on the molecular mechanisms of fiber development. But so far, key genes and regulatory mechanism involved in fiber growth are still unclear.Phytosterols are a large class of substances characterized by a tetracyclic skeleton, in which the content of sitosterol, campesterol and stigmasterol are high. Phytosterol is not only the precursor of plant hormone brassinosteroid but also one of the components of the plasma membrane. Phytosterol can regulate the fluidity and permeability of the plasma membrane. The phytosterols are supposed to play an important role in fundamental biological processes such as signal transduction, cellular sorting, cytoskeleton reorganization, asymmetric growth and infections diseases. The increase of plasma membrane area is very significant in the fiber growth process, it is one of the organelles changed dramaticly. Phytosterols that act as one membrane composition and a regulatory factor of membrane properties maybe play an important role in the fiber development. C-4demethylation is an important reaction in the synthesis of phytosterol, and performed by the C-4demethyl complex enzyme. To understand the effect and mechanism of phytosterols in fiber development, the3β-hydroxysteroid dehydrogenase (3β-HSD), a member of the complex genes was cloned from cotton fiber (Gh3β-HSD). Through sequence and expression pattern analysis, overexpressing Gh3β-HSD in tobacco and cotton and suppressing it in cotton, the functions of the Gh3β-HSD in cotton growth and development were analyzed, especially in the fiber growth and development. The following results are obtained:1. Gh3fl-HSD gene was cloned from upland cotton(Gossypium hirsutum. L)Based on the Arabidopsis At3β-HSD protein sequence, several cotton ESTs with high homologous to At3P-HSD were obtained by screening the cotton EST database, a consensus derived from these EST sequences contained a ORF, which encodes amino acid display high homologous with At3P-HSD. Sequence analysis showed that the sequence length is2061bp, containing a complete ORF a length of1683bp. The length of deduced amino acid sequence is560amino acid residues. The protein molecular weight is61.6KD, and its isoelectric point is9.256. The length of genomic sequence is5039bp, and the genomic sequence contains12exons and11introns. The deduced amino acid sequence is a membrance-protein, with3transmembrance domains. Gh3β-HSD has a high homology to other species’such as Arabidopsis, tobacco, poplar and rice. The homology with grape3β-HSD is75%, the similarity is86%; the homology with Arabidopsis3P-HSD is70%, the similarity is84%. Phylogenetic analysis showed cotton3β-HSD has a close relationship with the grape, poplar and ricinus communis3β-HSD. These results demonstrate that the cloned sequence is a homologue of3β-hydroxysteroid dehydrogenase gene, named Gh3β-HSD.2. The characteristics of Gh3β-HSD gene expressionThe expression pattern of a gene is an important evidence to analyse the gene function. To understand the function of Gh3β-HSD in cotton growth and development firstly, we detected the expression pattern of Gh3β-HSD by quatitative RT-PCR. The expression level of the gene is the highest in fiber, and then higher expression levels were detected in young stems, leaves and ovules than other tissues. Low expression levels were in the root, cotyledons and hypocotyls. The transcript of Gh3β-HSD was hardly detected in the stamen. The results indicated that Gh3β-HSD expression has tissue and organ specificity, predominantly accumulates in the fibers, ovules, young stems and leaves. Further analysis showed that the expression level of Gh3β-HSD in6DPA to20DPA fiber is higher than in the ovules at the same developmental stages as well as in the ODPA and2DPA ovule (including fiber). The results indicated that the gene is preferentially expressed in the fiber, closely related with fiber development. 3. Functional characterization of Gh3β-HSD promoter by transgenic tobaccoTo understand accurately the temporal and spatial expression pattern and the regulatory factors to affect Gh3β-HSD, the5’-upstream regulatory sequence of the gene whose length is1987bp was cloned. Sequence analysis showed that the sequence not only contains a number of light responsive elements and enhancer sequences, but also includes anaerobic response element, fungi response element, endosperm expression sequence, defense and stress response element, BRs response element, salicylic acid and ethylene response element. These indicated that the expression of Gh3β-HSD is affected by various factors. Then we carried out a5’deletion experiment of the promoter sequence. By using GUS gene as a report gene, the GUS activity was detected in various transgenic tobacco lines harbouring different length of promoter. The results showed that the904bp promoter sequence has no activity in transgenic tobacco. The1598bp promoter expressed highly in the epidermal hairs and vascular tissues of stem, and expressed in leaf epidermal hairs and mesophyll. Generally, the activity of the promoter was higher in young tissue than in old tissue. In roots Gh3β-HSD was mainly expressed in root tip, root hairs and the region of lateral root growth. At the same time, the activity of the promoter can be induced by salicylic acid in leaves and stems.4. Analysis the function of Gh3β-HSD gene by transgenic tobaccoIn order to understand the function of Gh3β-HSD as soon as possible, firstly, the Gh3β-HSD was expressed in transgenic tobacco. The results showed that expressing Gh3β-HSD in tobacco increased seed weight, and promoted seed germination. The plant height and root length increased in transgenic tobacco of T1generation. Furthermore, the number of lateral roots is increased dramatically in transgenic tobacco. At the same time, expressing Gh3β-HSD can improve the salt tolerance of tobacco. These results indicate that Gh3β-HSD is a functional sequence, and overexpressing Gh3β-HSD can affect the growth and development of the tobacco.5. Modifying the expression of Gh3β-HSD affect the growth of cotton plantsThrough the genetic transformation of cotton, transgenic plants identification and expression analysis, the transgenic cotton plants overexpressing and suppressing Gh3fl-HSD were obtained. Variation of traits showed that the leaf color of transgenic plants overexpressed Gh3β-HSD is dark green. There is no difference on plant shape between wildtype plants and transgenic plants in To generation. However, in T1generation, some plants overexpressing Gh3β-HSD have shortened branches since the internode was short. Compared with wildtype, the length of fruit stalk and the size of seed were larger, the boll was smaller, the number of seeds was fewer, and the fertility of pollen was lower in transgnic plants than wildtype. Contrarily, the transgenic cotton plants suppressing the expression of Gh3β-HSD were extremely short, and their leaves became smaller, internode become shorter, collateral became shorter. The number of epidermal hairs is significantly reduced in stem compared to wild-type cotton. Most transgenic plants can not flower, only few plants can flower, but all boll obscissed. These results indicate either overexpressing Gh3β-HSD gene or suppressing the expression of Gh3β-HSD affect the growth of cotton plants. It is suggested that Gh3β-HSD plays important roles in the growth and development of cotton plants.6. Modifying the expression of Gh3β-HSD affect the fiber growth and developmentOverexpressing Gh3β-HSD inhibited fiber cell initiation and elongation in transgenic cotton. There was no obvious difference about mature fiber length between transgenic cotton and wildtype in To generation. However, the fiber length of transgenic cotton was shorter than that of wildtype in T1generation. The fiber growth of transgenic cotton plants suppressing Gh3β-HSD was investigated in vitro culture system since the mature boll and fiber can not be gotten. The results showed that suppressing the expression of Gh3β-HSD also inhibits the growth of fiber. Similar result was obtained when the wildtype ovule was treated with trilostane, a specific inhibitor of3p-hydroxysteroid dehydrogenase. These results indicated that the Gh3β-HSD gene plays an important role in the growth and development of cotton fiber. |
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