Functions Of GhDET2 And GhKTN1 In Cotton Fiber Cell Development

Cotton is the world's leading fiber crop and is a mainstay of economy in China, which is the largest cotton producer and consumer around the world. Fiber is the major product of cotton production and the main raw material in textile industry. Due to the negative linkage between yield and quality of cotton fiber, it is very difficulty to synchronously improve both of them by traditional methods. With the advantages to overcome genetic obstacle among species and to transfer target genes purposively and rapidly, gene engineering provides an effective strategy to improve the yield and quality of cotton fiber. For this purpose, it is of significance to clone genes related fiber yield and quality and to elucidate the molecular basis of fiber development.Brassinosteroids (BRs) are a new class of phytohormones. Exogenous application experiments indicated that BRs can improve the yield and quality of cotton fibers. However, exogenous applications of BRs have instable effects on cotton growth, and are infeasible in field scale. Manipulating the levels of endogenous BRs in cotton by gene engineering provides us an effective and feasible strategy to improve the yield and quality of cotton fibers.Cytoskeleton plays an important role in growth and development of cotton fibers. Fiber elongation and secondary cell wall synthesis are closely related to the alignment pattern of cortical microtubule. At the elongation stage, orientation of cortical microtubule is transverse to the axis of cell elongation. At the stage of secondary cell wall synthesis, orientation of cortical microtubule is longitudinal to the axis of cell elongation. The microtubule alignment is regulated by microtubule disassembly factors and assembly factors. Given that cortical microtubule alignment is tightly consistent with the orientation of cellulose microfibril, which is a major factor determining the fiber strength, fiber elongation and secondary cell wall synthesis of cotton fibers and consequently the quality of cotton fibers can be modified by regulating the orientation of cortical microtubule alignment.To regulate the level of endogenous BRs and the alignment pattern of microtubule skeleton in fiber cells, the author cloned a steroid 5a-reductase gene (GhDET2) and a microtubule severing protein gene (GhKTN1) from cotton. The functions of these two genes had been investigated in transgenic tobacco and cotton. The main results are as follows.1. Cloning and expression analysis of GhDET2GhDET2 was cloned by 3'-RACE method on the basis of cotton ESTs homologous to Arabidopsis DET2. The cloned gene is 899 bp in length, including an ORF of 777 bp and 118-bp 3'-untranslated region and 4-bp 5'-untranslated region. The deduced GhDET2 protein contains 258 amino acids, and has a calculated molecular mass of 30kD. This protein is highly homologous to steroid 5α-reductases from pea, morning flower and Arabidopsis. A conserved domain in steroid 5α-reductases and also many important amino acid residues that are essential for the normal function of steroid 5a-reductase are conserved in GhDET2. No intron was found in the coding region of GhDET2. Southern blot analysis suggested that there existed a single copy of GhDET2 in the cotton genome and the GhDET2 gene may originate from D sub-genome in Gossypium genus. GhDET2 is highly expressed in cotton ovules and fibers. In various developmental stages of ovules and fibers, the expression level of GhDET2 keeps high at 0-DPA ovules (with fibers) to 13-DPA ovules (with fibers), and declines after 15-DPA. In various developmental stages of fibers, GhDET2 transcripts accumulate preferentially in fibers of rapid elongation stage. Furthermore, the expression levels of GhDET2 were always higher in ovules (with fibers) of cv. Xuzhou 142 than its fuzzless-fiberless mutant. These results suggested that the GhDET2 may play an important role in cotton fiber growth and development.2. Biochemical function of GhDET2To determine whether GhDET2 encodes a functional steroid 5a-reductase, the coding region of GhDET2 was cloned into a mammalian expression vector, pcDNA3.1. The resulting plasmid was introduced into Chinese hamster ovary (CHO) cells to measure steroid 5a-reductase activity. Using progesterone, a substrate of steroid 5α-reductases to assay the enzyme activity in the gene expression product, the result showed that 5α-reductase activity was exhibited in the transgenic CHO cells and the activity was inhibited by finasteride, a specific inhibitor of steroid 5α-reductase, demonstrating that the GhDET2 encodes a functional steroid 5α-reductase.3. Function analysis of GhDET2 in transgenic tobaccoTo analyze the biological functions of GhDET2, this gene was first over-expressed in tobacco. Transgenic tobaccos over-expressing GhDET2 had an increased biomass with both vegetative and productive growth enhanced. Compared to wildtype plants, the seed weight, hypocotyl length, and root length of transgenic tobacco were 53.1%, 80%, and 190% increased respectively. Over-expression of GhDET2 promoted the initiation and growth of lateral roots, adventitious roots and root hairs. In addition, the root tip of transgenic seedlings was more sensitive to gravity than that of wildtype. These phenotypical variations in transgenic tobacco are similar to these results from BL exogenous application. On the contrary, finasteride, a specific inhibitor of steroid 5α-reductase, inhibits the growth of root and hypocotyl of wildtype and transgenic tobaccos. These results indicated that over-expression of GhDET2 can increase the endogenous BR content in transgenic tobaccos.The relationship of BRs to various phytohormones was further investigated using transgenic tobaccos over-expressing GhDET2. Auxins (IAA and IBA) shew opposite effects on the growth of root and hypocotyls in the over-expressing GhDET2 background. IAA and BRs independently promote hypocotyl elongation, but counteract in promoting root growth, while IBA and BRs counteract in promoting hypocotyls elongation, but enhance root growth independently. BRs and cytokinin act independently on root growth, while these two kinds of hormones counteract in promoting hypocotyls growth. BRs and gibberellin enhance root growth synergistically, but display some counteraction in promoting hypocotyls elongation. Finally, BRs exhibit counteraction to abscis acid on seed germination and root growth, and these two hormones act relatively independently on the growth of hypocotyls.4. GhDET2 plays important roles in cotton fiber initation and elongationTo further elucidate the functions of the GhDET2 in cotton development, particularly in fiber development, transgenic cottons with GhDET2 overexpressed or antisense suppressed were obtained. Morphologically, the antisense GhDET2 plants were much shorter than the control plants that were transformed with an empty vector. The antisense GhDET2 plants exhibited reduced leaf size as well as shortened internodes and branches. The pollen grains from antisense plants were sterile. Cotton bolls produced from the antisense GhDET2 plants aborted within 3 DPA to 5 DPA, even if the plants were pollinated with wildtype pollens. Sense GhDET2 transgenic plants displayed almost normal growth. However, the branches of GhDET2 overexpressing plants were shorter, and the size of the main stem leaf was smaller than controls. Similar to antisense plants, pollens from sense GhDET2 transgenic cottons were sterile and boll drop occurred within a few days after anthesis. By the scanning electron microscopy, on the day of anthesis, the density of fiber initials on an antisense GhDET2 plant ovule was greatly reduced relative to a control plant ovule. On average, there were approximately 1,600 fiber cells per ovule in antisense GhDET2 plants, much less than the fibers in the control ovules (approximately 5,600 fibers per ovule). Many fiber cells from the ovules of the antisense GhDET2 plant appeared smaller than those on ovules of control plants. Fiber cell elongation was also inhibited in plants expressing antisense GhDET2. Through ovule culture in vitro, fiber cell elongation was significantly reduced in the antisense GhDET2 ovules. However, addition of 100 nM 24-epibrassinolide (EBL, an biologically active BR) to the culture medium restored fiber cell elongation in antisense GhDET2 plant ovules near to wild type levels. Also, finasteride, an inhibitor of steroid 5α-reductase, severely inhibited the elongation of cotton fibers. Ovules from wild type 2-DPA plants, a time when many fiber cells have already initiated, were cultured for 5 days on the medium containing different concentrations of finasteride. Higher finasteride concentrations produced even greater inhibition of fiber elongation. Little fiber cell elongation was observed in ovules cultured for 5 or 15 days with 159μM finasteride. Finasteride-mediated inhibition of fiber elongation was partially overcome by 100 nM EBL.5. Ectopic expression of GhDET2 under control of a seed coat specific promoter, PFBP7, increases fiber number and fiber lengthA petunia-derived seed coat-specific gene (FBP7) promoter, PFBP7, was used to study the effects of GhDET2 on fiber initiation and elongation in the absence of developmental defects. As expected, all transgenic plants harboring the PFBP7::GhDET2 cassette had normal morphology and growth. Fiber number and fiber length were increased in the resulting transgenic T2 lines, for example, the fiber number and the fiber length of the F2-7-10 transgenic line were 6240±610 and 32.98±0.85mm respectively, a 22.6% and 10.7% increase relative to control plants (5090±380 fibers and 29.78±1.10mm fiber length. However, no obvious improvement of fiber number and length was observed in transgenic lines in which no obvious change of GhDET2 transcript was detected. These results revealed that GhDET2 and BRs play an important role in the fiber cell initiation process and in fiber cell elongation.6. Cloning and expression analysis of GhKTN1To clone cotton microtubule severing protein gene, a partial cDNA was firstly amplified from fibers on the basis of cotton ESTs homologous to Arabidopsis microtubule severing protein. The full-length ORF sequence of GhKTN1 was obtained by genomic DNA walking. The GhKTN1 ORF is 1563 bp in length, and encodes a protein of 520 amino acids. The deduced GhKTN1 protein shares high homology with microtubule severing proteins from Arabidopsis, rice and human. Multiple alignments revealed that GhKTN1 contained an AAATPase domain, which is conserved in other microtubule severing proteins. The predicted three-dimensional structure of GhKTN1 was similar to that of Arabidopsis microtubule severing protein. These analyses suggested that the cloned GhKTN1 is a homologous gene of microtubule severing proteins. Expression analysis was performed with quantitative real-time RT-PCR. In all detected tissues and organs, the highest expression level of GhKTN1 was found in ovules and fibers. Its expression levels in roots, hypocotyls and cotyledons were higher than those in leaves and 0-DPA flowers. At various developmental stages of cotton fibers, the expression level of GhKTN1 is low from 5-DPA to 13-DPA, remarkably increases at 16-DPA, and rises continuously hereafter. These results indicated that GhKTN1 gene may have a role in the set of secondary cell wall synthesis and secondary cell wall biosynthesis.7. GhKTN1 influences the set of secondary cell wall biosynthesis and increases cell wall thickness and fiber strengthTo further clarify the function of GhKTN1 in the development of cotton fibers, the sense and antisense GhKTN1 were constitutively expressed in transgenic cottons. The fiber length of transgenic cottons overexpressing GhKTN1 gene is 29.8% shorter than that of wildtype fibers. The thickness of secondary cell wall of cotton fibers and the micronaire value of cotton fibers increased in transgenic plants overexpressing GhKTN1. The fiber strength of sense GhKTN1 fibers increased, 19.5% higher than that of wildtype fibers. The expression level of GhKTN1 is suppressed in antisense GhKTN1 cottons, which results in a thinner fiber secondary cell wall and a lower fiber strength (7.8% decreased relative to wildtype). However, no obvious differences were observed on the fiber length, fiber shape and micronaire value between the antisense GhKTN1 and wildtype plants. These results demonstrated that the GhKTN1 gene plays an important role in the development of cotton fibers, particularly, in the secondary cell wall formation of cotton fibers. Microtubule alignment pattern and cellulose microfibril orientation were investigated microscopely. Remarkable changes of microtubule alignment pattern in transgenic plants expressing sense and antisense GhKTN1 were observed. Increasing the expression level of GhKTN1 promoted the microtubule dynamics. This consequently speeded up the cellulose synthesis in fibers and led the secondary wall accumulation time 3 days earlier than that of the control. The study provides a new stretagy to improve fiber strength and fiber length by modulating the expression of genes involved in microtubule dynamics in cotton fibers.