Impacts Of Suppressing GhUPL7 Gene On Ovule And Trichome Development In Cotton

UPL7 is a class of HECT, homologous to the E6-associated protein carboxyl terminus, ubiquitin protein ligase which specifically recognizes the target proteins of ubiquitination pathway which is intimately related to plant growth and development. To unravel the function and mechanism of UPL7 in cotton fiber development, previously GhUPL7 gene was cloned from cotton. The text identified firstly HECT gene in upland cotton and analyzed structure characteristics of HECT gene of the cotton genomes. Then, We further detected expression pattern of GhUPL7 and expression characteristics of upstream promoter sequences in plants. By detecting and observing change of ovules, fibers and leaf trichomes in suppression transgenic plants made it clear that the GhUPL7 affect growth and development on the cotton part organ. Detailed results are as follows: 1. Phylogenetic analysis of HECT gene in upland cottonCloned GhUPL7 gene shared the similarity with 99 % Gh_A01G1811 and 98 % Gh_D01G2049 two genes from upland cotton. There are 25 homologous genes encoding HECT protein sequence in cotton, namely HECT genes. These 25 upland cotton HECT gene performed phylogenetic analysis with UPLs in Arabidopsis, GhUPLs can be divided into four branches, namely subfamily I, II, III and IV. Gh_A01G1811 and Gh_D01G2049 have become closer genetic relationship with Arabidopsis AtUPL7 belonged to the subfamily IV. To better understand the differences of HECT genes structure, compared to exon-intron structure of the upland cotton genome sequence and HECT protein domain framework according to their phylogenetic relationships. The results showed that the N-terminus contains other domains that tipped the balance HECT protein interaction with different substrates except C-terminus HECT domain of upland cotton HECT gene. HECT subfamily IV N-terminus domain has inexistence or unknown domain. Intimate relationship of HECT gene is much more similar in the gene structure, especially on the number of exons and introns, the main difference is their respective exon-intron length. The above analysis shows GhUPL7 is actually HECT type ubiquitin protein ligase, and conserved in evolution. 2. Analysis of GhUPL7 expression patternDesigned GhUPL7 specific Real-Time PCR primers to detect GhUPL7 gene expression patterns in different organs indicated its relative higher expression levels of stigma, but low in other organs. Detection GhUPL7 gene expression patterns in different periods of cotton development showed fiber elongation rapidly expression levels are relatively higher. 3. Cloning and function analysis of GhUPL7 upstream sequenceAnalysis upstream regulatory sequence of GhUPL7 gene by PlantCARE website, we found that the regulatory sequences had multiple cis-acting elements divided into six classes according to the function, namely light effects associated elements, hormone response related components, tissue-specific expression elements, stress response related components, as well as binding sites and unknown functional elements. These cis-acting response elements were associated with the light.GhUPL7 upstream sequence was cloned and transformed into tobacco. Tobacco GUS staining showed GUS gene driven by GhUPL7 promoter is expressed mainly in the tobacco stigma, and secondly in the receptacle, ovule surface. Further detection GUS activity in different tissues of GhUPL7 promoter transgenetic tobacco, enzyme activity was the strongest in stigma, but only 76.39 nmol/min/mg, and relatively low enzyme activity in different periods of receptacle and ovary. GUS activity of receptacle and ovary in 8 DPA respectively were 11.13 nmol /min/mg and 10.28 nmol / min / mg.These results suggested that GhUPL7 promoter direct GUS activity mainly in the floral organs. 4. Seeds enlarged and seed index increased of antisense inhibition of GhUPL7 geneGhUPL7 is a specific enzyme in ubiquitination reaction that are closely related to plant growth and development. To investigate this gene whether was involved in three major nutrients metabolic pathway by ubiquitination, plant expression vector CaMV35S::antisenseGhUPL7 was constructed, and introduced into upland cotton(J14), Real-Time PCR screening obtained antisense inhibition GhUPL7 transgenic plants by designed RT primers. Seeds were harvested and screened T1 generation six transformants 42 plants were got. Total nitrogen and total soluble sugar content of 10 DPA ovule were measured. The results showed that the highest total nitrogen content increased by 26.60 %; the highest total soluble sugar content increased by 32.96 %. Corresponding seeds of formation from ovule enlarged, which was consistent with Arabidopsis mutant upl7 phenotype. Yield traits implyed that seed index had an increase of up to 14.86 %. We infered GhUPL7 gene may be involved in the ubiquitin-proteasome pathway, affecting the carbon and nitrogen metabolism. 5. Fuzz of antisense inhibition GhUPL7 transgenic plants increased in number, lint barely changedFiber nutrition is supplied by the ovule. In order to examine influence of antisense inhibition GhUPL7 gene to fiber development, fiber initiation and mature fiber phenotype of antisense inhibition GhUPL7 T1 generation plants were observed, sugar content was measured, and yield traits were statistically analyzed. Antisense inhibition of GhUPL7 gene had impact on cotton fiber initiation observed by SEM. Results showed that fiber cell initiation appear to abnormal fibers, but the number of fiber spike had no significant changes. Measurement of sugar content displayed antisense inhibition GhUPL7 plant fiber total soluble sugar decreased by 30.66 %; mature fiber observation showed that antisense inhibition GhUPL7 lint dischanged or slightly decreased, but fuzz increased in number. Yield traits results showed lint did not change significantly, the highest decreased by 7.16 %. 6. Antisense inhibition GhUPL7 gene increased cotton leaf trichome branchesStudies reported UPL3 of UPLs family in Arabidopsis related to the development of trichome, therefore leaves trichomes of antisense inhibition GhUPL7 the same as HECT family were observed and counted. The results showed the percentage of the number of branches 1-3 reduced significantly and 4-5 branches increased significantly. This result was consistent with phenotype of Arabidopsis mutant upl7 trichome branches number. Antisense inhibition GhUPL7 gene increased cotton leaf trichomes branches number, we deduced trichomes and fiber fuzz share a similar mechanism.