Isolation Of Mesophyll Protoplast And Establishment Of Gene Transient Expression System In Cotton And Preliminary Function Analysis Of The Gene Containing DUF761 DOMAIN

1. Cotton is important economic crop. With cotton genome sequence information released, functional analysis of a large number of cotton gene related to important traits need to be implemented. At present, the diploid G. raimondii genome sequence has been released. To construct cotton protoplasts gene transient expression system will accelerate the process of cotton functional genomics research.This paper aimed to set up a stable and efficient transient expression system based on cotton mesophyll protoplasts produced from cotyledon. We analyzed the key factors related to isolating effectively cotton mesophyll protoplasts and used it as a vehicle to express transfected genes for functional studies. The main research results are as follows:1) The isolation of cotton mesophyll protoplast. Take cotton protoplast as receptors, the cotton mesophyll protoplast system is optimized in three aspects, the leaf age, osmotic concentration and the digestion time, and the enzymatic method is used to isolate mesophyll protoplasts. The major technical system includes:healthy 12-day-old young cotton cotyledons in natural growth condition were selected as the explant, and were digested with enzyme solution including 1.5% cellulose,0.4% macerozyme,0.5 mol L-1 mannitol,20 mmol L-1 KCl,20 mmol L-1 MES,0.1 mol L-1 CaCl2, and 1.0 g L-1 BSA for eight hours with gentle shaking at 26℃ and under dark conditions.The concentration of the pure cotton mesophyll protoplasts was more than 1.0×106/ml, and the protoplast activity more than 90%.2) The transient transformation expression system of cotton mesophyll protoplast. Using the system, we integrated a cotton zinc finger protein gene GhZFP2 into pJIT166-GFP plasmid vector, constructed the GhZFP2:GFP fusion vectors, and transformed it into purified cotton mesophyll protoplast mediated with 40% PEG4000. As a result, cotton mesophyll protoplast with high transformation frequency was obtained. Transient expression detection for GhZFP2 protein showed that it was clearly located in the cell nucleus.2. Cotton fiber is the longest single cell, and fiber development has the similar mechanism with that in Arabidopsis thaliana hairs. A gene with DUF761 DOMAIN was cloned from fiber development library of upland cotton in our laboratory early days. It encoded 330 amino acids with two copies in the tetraploid cotton, with each in A subgenome and D subgenome, of which the theorical pI of At copy is 7.77 and its molecular weight (MW) is 37.60 KDa, and the theorical pI of Dt copy is 6.29 and its molecular weight (MW) 37.63 KDa. It has a highter expression in root, stem and different periods of fiber development period. In order to further clarify its functional role, an analytical study of the subcellular location and its transgenic function is conducted. The location of the protoplast subcellular in cotyledon shows that two copies of A and D subgroup of the gene are both located in the chloroplast. The over-expressed vectors of At (Dt) gene were transformed into the Arabidopsis thaliana genome by the Agrobacterium-mediated transformation, and the PCR analysis of transformed exogenous gene shows that eight transgenic pure lines with DUF761At and ten transgenic pure lines with DUF761Dt are obtained. The analysis of RT-PCR reveals that the expression level of the A4, A7, A8, D1, D2 and D4 transgentic strains is much higher than the wild type. The phenotypic analysis proves that compared with WT, the transgenic Arabidopsis that has been transferred into the two sets of DUF761At (Dt) geng shows obviously different phenotype. Without resistance, the length of root has no difference between DUF761At and the wild type, but its branch number was significantly more than the wild type; while the DUF761Dt has no main root and more branch root, and its root is significantly shorter than the wild type. However, under the resistance condition, the root of transgenic DUF761At hovered over the medium, which is unable to go through the solid medium, while, the root of transgenic DUF761Dt is significantly shorter than the wild type, and could enter the solid medium. It showed that functional differentiation occurs in the two homologous genes in the control of root hair development.