海岛棉GbHCT13基因的功能验证

该研究利用海岛棉‘新海21’和陆地棉ND203以及模式植物拟南芥,通过转基因及荧光定量检测等方法探究海岛棉GbHCT13基因(GenBank 登录号MW048849)在纤维发育中的功能。结果显示：(1)成功构建重组载体pCAMBIA3301 GbHCT13,经农杆菌介导法转化、除草剂抗性基因筛选、荧光定量检测方法鉴定获得转GbHCT13基因拟南芥T₃代植株4株;qRT PCR检测表明,转基因植株中GbHCT13基因表达量较野生型极显著增加。(2)转基因拟南芥过表达GbHCT13基因使植株同一时期的生长较野生型旺盛,株形、叶片数、抽薹数和茎秆表皮毛数量均与野生型存在差异;组织化学分析发现,转GbHCT13基因的拟南芥较野生型茎秆初生木质部生长活跃,导管增粗,次生木质部导管细胞壁横截面积变大,但髓质细胞无明显变化;过表达GbHCT13使拟南芥中木质素合成途径基因发生不同程度改变,其中CAD、CCoAOMT、PAL和4CL与GbHCT13基因的表达呈正相关。(3)经大田筛选、分子鉴定,成功获得转GbHCT13基因棉花植株3株;转GbHCT13基因棉花的棉纤维伸长率增加,纤维强度增大;沉默GbHCT13基因使棉花植株木质素含量降低,茎秆表皮毛数量减少,木质部导管细胞数量减少,导管细胞壁中木质素沉积量降低,而棉株并未发生株高上的明显矮化现象,且木质素合成通路中的CAD、CCoAOMT、CCR、PAL 4个基因的表达均呈降低趋势,说明抑制GbHCT13使得棉花生长代谢受阻,影响纤维发育起始。研究表明,GbHCT13基因能影响棉花植株中木质素合成从而调控纤维的生长发育,其功能与GbHCT13基因在模式植物拟南芥中的基本一致。     

Cotton (Gossypium hirsutum) 14‐3‐3 proteins participate in regulation of fibre initiation and elongation by modulating brassinosteroid signalling

Cotton (Gossypium hirsutum) fibre is an important natural raw material for textile industry in the world. Understanding the molecular mechanism of fibre development is important for the development of future cotton varieties with superior fibre quality. In this study, overexpression of Gh14‐3‐3L in cotton promoted fibre elongation, leading to an increase in mature fibre length. In contrast, suppression of expression of Gh14‐3‐3L, Gh14‐3‐3e and Gh14‐3‐3h in cotton slowed down fibre initiation and elongation. As a result, the mature fibres of the Gh14‐3‐3 RNAi transgenic plants were significantly shorter than those of wild type. This ‘short fibre’ phenotype of the 14‐3‐3 RNAi cotton could be partially rescued by application of 2,4‐epibrassinolide (BL). Expression levels of the BR‐related and fibre‐related genes were altered in the Gh14‐3‐3 transgenic fibres. Furthermore, we identified Gh14‐3‐3 interacting proteins (including GhBZR1) in cotton. Site mutation assay revealed that Ser163 in GhBZR1 and Lys51/56/53 in Gh14‐3‐3L/e/h were required for Gh14‐3‐3‐GhBZR1 interaction. Nuclear localization of GhBZR1 protein was induced by BR, and phosphorylation of GhBZR1 by GhBIN2 kinase was helpful for its binding to Gh14‐3‐3 proteins. Additionally, 14‐3‐3‐regulated GhBZR1 protein may directly bind to GhXTH1 and GhEXP promoters to regulate gene expression for responding rapid fibre elongation. These results suggested that Gh14‐3‐3 proteins may be involved in regulating fibre initiation and elongation through their interacting with GhBZR1 to modulate BR signalling. Thus, our study provides the candidate intrinsic genes for improving fibre yield and quality by genetic manipulation.     

Functional characterization of <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> profilin 1 gene (<Emphasis Type="Italic">GhPFN1</Emphasis>) in tobacco suspension cells

Cotton fiber is an extremely long plant cell. Fiber elongation is a complex process and the genes that are crucial for elongation are largely unknown. We previously cloned a cDNA encoding an isoform of cotton profilin and found that the gene (designated GhPFN1) was preferentially expressed in cotton fibers. In the present study, we have further analyzed the expression pattern of GhPFN1 during fiber development and studied its cellular function using tobacco suspension cells as an experimental system. We report that expression of GhPFN1 is tightly associated with fast elongation of cotton fibers whose growth requires an intact actin cytoskeleton. Overexpression of GhPFN1 in the transgenic tobacco cells was correlated with the formation of elongated cells that contained thicker and longer microfilament cables. Quantitative analyses revealed a 2.5–3.6 fold increase in total profilin levels and a 1.6–2.6 fold increase in the F-actin levels in six independent transgenic lines. In addition to the effect on cell elongation, we also observed delayed cell cycle progression and a slightly lower mitotic index in the transgenic cells. Based on these data, we propose that GhPFN1 may play a critical role in the rapid elongation of cotton fibers by promoting actin polymerization. Hai-Yun Wang and Yi Yu contributed equally to this work.     

The R3-MYB Gene GhCPC Negatively Regulates Cotton Fiber Elongation

Cotton (Gossypium spp.) fibers are single-cell trichomes that arise from the outer epidermal layer of seed coat. Here, we isolated a R3-MYB gene GhCPC, identified by cDNA microarray analysis. The only conserved R3 motif and different expression between TM-1 and fuzzless-lintless mutants suggested that it might be a negative regulator in fiber development. Transgenic evidence showed that GhCPC overexpression not only delayed fiber initiation but also led to significant decreases in fiber length. Interestingly, Yeast two-hybrid analysis revealed an interaction complex, in which GhCPC and GhTTG1/4 separately interacted with GhMYC1. In transgenic plants, Q-PCR analysis showed that GhHOX3 (GL2) and GhRDL1 were significantly down regulated in −1–5 DPA ovules and fibers. In addition, Yeast one-hybrid analysis demonstrated that GhMYC1 could bind to the E-box cis-elements and the promoter of GhHOX3. These results suggested that GhHOX3 (GL2) might be downstream gene of the regulatory complex. Also, overexpression of GhCPC in tobacco led to differential loss of pigmentation. Taken together, the results suggested that GhCPC might negatively regulate cotton fiber initiation and early elongation by a potential CPC-MYC1-TTG1/4 complex. Although the fibers were shorter in transgenic cotton lines than in the wild type, no significant difference was detected in stem or leaf trichomes, even in cotton mutants (five naked seed or fuzzless), suggesting that fiber and trichome development might be regulated by two sets of genes sharing a similar model.     

Transgenic cotton expressing CYP392A4 double‐stranded RNA decreases the reproductive ability of Tetranychus cinnabarinus

As a polyphagous pest, Tetranychus cinnabarinus has the ability to overcome the defense of various hosts, and causes severe losses to various economically important crops. Since the interaction between pest and host plants is a valuable clue to investigate potential ways for pest management, we intend to identify the key genes of T. cinnabarinus for its adaption on cotton, then, with RNA interference (RNAi) and transgenic technology, construct a transgenic cotton strain to interfere with this process, and evaluate the effect of this method on the management of the mites. The difference of gene expression of T. cinnabarinus was analyzed when it was transferred to a new host (from cowpea to cotton) through high‐throughput sequencing, and a number of differentially expressed genes involved in detoxification, digestion and specific processes during the development were classified. From them, a P450 gene CYP392A4 with high abundance and prominent over‐expression on the cotton was selected as a candidate. With transgenic technology, cotton plants expressing double‐stranded RNA of CYP392A4 were constructed. Feeding experiments showed that it can decrease the expression of the target gene, result in the reduction of reproductive ability of the mites, and the population of T. cinnabarinus showed an apparent fitness cost on the transgenic cotton. These results provide a new approach to restrict the development of mite population on the host. It is also a useful attempt to control piercing sucking pests through RNAi and transgenic technology.     

Cotton PRP5 gene encoding a proline-rich protein is involved in fiber development

Proline-rich proteins contribute to cell wall structure of specific cell types and are involved in plant growth and development. In this study, a fiber-specific gene, GhPRP5, encoding a proline-rich protein was functionally characterized in cotton. GhPRP5 promoter directed GUS expression only in trichomes of both transgenic Arabidopsis and tobacco plants. The transgenic Arabidopsis plants with overexpressing GhPRP5 displayed reduced cell growth, resulting in smaller cell size and consequently plant dwarfs, in comparison with wild type plants. In contrast, knock-down of GhPRP5 expression by RNA interference in cotton enhanced fiber development. The fiber length of transgenic cotton plants was longer than that of wild type. In addition, some genes involved in fiber elongation and wall biosynthesis of cotton were up-regulated or down-regulated in the transgenic cotton plants owing to suppression of GhPRP5. Collectively, these data suggested that GhPRP5 protein as a negative regulator participates in modulating fiber development of cotton.     

Genetic mapping and comparative analysis of seven mutants related to seed fiber development in cotton

Mapping of genes that play major roles in cotton fiber development is an important step toward their cloning and manipulation, and provides a test of their relationships (if any) to agriculturally-important QTLs. Seven previously identified fiber mutants, four dominant (Li ₁, Li ₂, N ₁ and Fbl) and three recessive (n ₂, sma-4(h _a), and sma-4(fz)), were genetically mapped in six F₂ populations comprising 124 or more plants each. For those mutants previously assigned to chromosomes by using aneuploids or by linkage to other morphological markers, all map locations were concordant except n ₂, which mapped to the homoeolog of the chromosome previously reported. Three mutations with primary effects on fuzz fibers (N ₁, Fbl, n ₂) mapped near the likelihood peaks for QTLs that affected lint fiber productivity in the same populations, perhaps suggesting pleiotropic effects on both fiber types. However, only Li ₁ mapped within the likelihood interval for 191 previously detected lint fiber QTLs discovered in non-mutant crosses, suggesting that these mutations may occur in genes that played early roles in cotton fiber evolution, and for which new allelic variants are quickly eliminated from improved germplasm. A close positional association between sma-4(h _a ), two leaf and stem-borne trichome mutants (t ₁ , t ₂), and a gene previously implicated in fiber development, sucrose synthase, raises questions about the possibility that these genes may be functionally related. Increasing knowledge of the correspondence of the cotton and Arabidopsis genomes provides several avenues by which genetic dissection of cotton fiber development may be accelerated.     

Role of abscisic acid in cotton fiber development

Fibers of three cotton cultivars varying widely in their final fiber length, i.e., long staple (Gossypium hirsutum H-4), middle staple (G. Hirsutum H-8), and short staple (G. Arboretum G. Cot-15) were analyzed to study the role of ABA in fiber elongation and dry matter accumulation, in vivo and in vitro. The fibers were analyzed for different growth parameters and endogenous ABA content during the entire period of their development using indirect ELISA by raising the antibodies against ABA. From growth analysis, cotton fiber development was divided into four distinct phases, (i) initiation, (ii) elongation, (iii) secondary thickening, and (iv) maturation. An inverse correlation between final fiber length and ABA content was observed in all the cultivars. In long staple cultivar (H-4), rapid ABA accumulation started after fiber had attained peak elongation growth while, in short staple cultivar (G. Cot-15), ABA accumulation was observed even during elongation growth. Significant inhibition in length of short and middle staple cultivars as compared to long staple cultivar was observed in in vitro grown fibers when media were supplemented with ABA (1, 3, and 5 mg/l). The addition of growth promoters like NAA and GA, along with ABA, has reduced the inhibition in fiber elongation in all the cultivars. These results suggest a regulatory role of ABA in cotton fiber elongation along with auxins and gibberellins. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 1, pp. 68–74. The text was submitted by the authors in English.