Cloning, Characterization And Functional Analysis Of A Putative Receptor-Like Kinase That Is Involved In Drought, Salt And Verticillium-Tolerance In Cotton

Cotton (Gossypium spp.) is an important economic crop and the largest source of textile fiber in the world. As environmental degradation, cotton in cotton growing areas is subject to extreme drought salinity and temperature and diseases such as the invasion of diseases, particularly Verticillium wilt of cotton, which is known as cancer in cotton production, each of which can impede cotton growth and production. Gossypium hirsutum is the major cotton species planted in China and breeding to develop fusarium-resistant and fine fiber quality cultivars has been very fruitful, however, breeding to develop cultivars resistant to V. dahliae has proven to be unsuccessful, largely due to the lack of germplasm known to be immune or highly resistant to the fungal pathogen V. dahliae. Therefore, through research on stress resistance mechanism of cotton, and the use of genetic engineering methods to cultivate fine cotton varieties will be the main direction of future cotton breeding. The receptor-like kinase (RLK) belongs to a subgroup of kinase with at least 600 members in the Arabidopsis genome, Some of them,for example BRI1,CLAVATA1,Pto,SRK,Xa21 and so on, participate in a diverse range of processes, including self-versusnonself recognition, disease resistance, stress resistance, regulation of development, and hormone perception, but much less is known about their functions.Gossypium barbadense L. cv. Hai7124, a widely used Verticillium wilt-resistant cultivar in China for genetic study and breeding, was employed for cloning the receptor-like kinase, which has a 1,080 bp ORF, coding 360 amino acids, without intron. It was homologous with receptor-like protein and included 11 conserved domains of receptor-like protein, so we named the gene as GbRLK. The RT-PCR and Northern blotting results indicated that the expression of GbRLK was induced at time point 96 h after inoculation used V. dahliae. Southern blotting result confirmed at least three copies in Hai7124 genome. We amplified the genes from two diploid cottons, and the A sub-genome and the D sub-genome in tetraploid cotton, genomic sequences analysis showed that a base was deleted in the sequences of GbRLK in the A-genome diploid cotton and the At sub-genome in tetraploid cotton, which lead to premature termination, so we concluded that the functional GbRLK evolved from the Dt sub-genome in tetraploid cotton, and it became another gene or a unfunctional gene in At sub-genome. We constructed vector fused with GFP report gene and transformed it into the epidermal cells of onion via gene gun. It is found that the the GFP was assembled in cellular membrane, and not in the cellular wall by treatment of 20% sucrose. For further investigation the expression characteristics of GbRLK, we treated the Gossypium barbadense L. cv. Hai7124 with ABA, PEG, NaCl, and low temperature. The expression of GbRLK was induced after treatment by ABA, PEG and NaCl, rather than by low temperature. With the gene location and expression of the stress treatment, it is supposed that the gene should function as the signal transduction.To further verify the function of GbRLK, we constructed an overexpression transformation vector drived by the constitutive CaMV35S promoter in pBI121 backbone structure, and transformed it into Arabidopsis thaliana ecotype Col-0. Based on pedigree selection of Km resistance, we obtained four homozygous transgenic lines. Southern blotting analysis showed that these four transgenic pure lines contained one to three copies, respectively.The sensitivity of the ABA, and the increased resistance to drought, high salinity and the Verticillium dahliae were observed in these transgenic GbRLK Arabidopsis thaliana lines. The germination rate of transgenic Arabidopsis thaliana was significantly inhibited in the medium contained different concentration of ABA. On the MS medium containing 100、200、400、800nmol/L ABA, the germination rate of the transgenic line K6 which was less sensitivity in four transgenic lines was 88.36%,78.66%,56.25%, and 27.08%, the germination rate of the transgenic line K2 were 76.24%,68.14%,18.35%, 2.17%, which was the most sensitivity in four transgenic lines. However, the germination rate of the wild-type was 92.73%,86.59%,80.41%, and 68.19%. After six weeks growth on medium contained different concentration of mannitol and sodium chloride, the survival rate of transgenic plants was significantly higher than that of non-transgenic plant. On medium contained 100 mM NaCl, compared with 22.9% of the wild-type, the survival rate of transgenic lines K2, K3, K5, and K6 was 62.9%,43.9%,45.8%, and 37.5%, respectively. On medium contained 500 mM and 750 mM mannitol, the survival rate of transgenic lines k6 which has less resistance in four transgenic lines was 51.3% and 38.3%, however, the survival rate of the wild-type was 33.1% and 21.1%. Compared the water loss rate of leaves in vitro between the transgenic and non-transgenic Arabidopsis thaliana,we found the water loss rate of the transgenic lines K2, K3, K5, and K6 was 15.9%,19.2%,17.9%, and 20.8% after 60min, respectively, however the wild-type was 24.1%. At the time point 120 min, the difference began reaching to the highest between the transgenic and non-transgenic Arabidopsis thaliana. At the time point of 360 min, the water loss rate of transgenic line K6 was 64.2% when compared with the wild-type (81.9%). Transgenic Arabidopsis thaliana lines have produced Verticillium resistance after inoculated with the defoliating strain VD8 and non-defoliating strain Bp2. Transgenic and non-transgenic Arabidopsis showed significant differences, fresh weight, anthocyanin accumulation, chlorophyll content, disease stage, incidence of the transgenic line K6 was 0.44g,17.34 nmol/g,0.74 nmol/g, 2.0, and 80% compared with the non-transginc 0.24g,28.88 nmol/g,0.49 nmol/g,3.0, and 93.4%, respectively. We found that the proliferation of Verticillium dahliae was inhibited in transgenic Arabidopsis thaliana by use of qRT-PCRIn order to reveal the mechanism of GbRLK gene for improving the tolerance of stress resistance in transgenic Arabidopsis thaliana, we studied the gene expression profile of transgenic GbRLK and non-transgenic Arabidopsis thaliana including two genetically modified pure clones (K6 and K2) with differences in resistance to Verticillium, and the non-transgenic receptor Col-0, respectively. We found the overexpression of GbRLK in transgenic GbRLK Arabidopsis thaliana prompted the changes of many genes expression level. By comparative analysis of genes expression level in two genetically modified pure clones and the non-transgenic Col-0, we find 809 different expression genes including 347 up-regulated genes and 462 down-regulated genes between them. Among them,18 up-regulated genes, incuding 6 transcription factors, bHLH100, bHLH038, bHLH101, bHLH039, RRTF1, and CBF2, were specially expressed in transgenic plants before inoculation used V. dahliae. In the up-regulated genes, there were many transcription factors, DNA binding proteins, electron carrier activity, carbohydrate transporter activity, acid phosphatase activity, copper ion binding and so on, and transcription factors and DNA binding proteins were the largest proportion, up to 10%. Meanwhile, many associated with stress, but there were few genes which were proved playing a key role in disease resistance. It suggested that GbRLK genes be involved the biological and abiotic stress responses which were regulated by ABA signaling. This result is foundation for in-depth study GhRLK gene resistance mechanism and the construction of plant disease resistance signaling network.We further cloned approximately 3,200 bp promoter sequence on 5 ’upstream of this gene via the Sefa-PCR. Sequence analysis revealed that the region contains many cis-acting stress response elements such as ABRE, W-Box, MYB-core, W-Box core, TCA-element and so on. Based on the results, we chosen the 1,890 bp sequence on its 5’upstream to construct three missing expression vectors and transformed them into Arabidopsis thaliana. GUS histochemical staining analysis showed that the gene expressed mainly in the roots, vein, and the catheter, and a slight expression in stomatal. The GbRLK promoter activity can be induced by treatment of ABA, PEG, NaCl and Verticillium dahliae in transgenic Arabidopsis thaliana. Impressively, deletion analysis further revealed that the promoter segments from-1 to-664bp contained novel cis-elememt like ABRE, MYB-core, W-Box core in response to ABA, PEG, NaCl, and Verticillium dahliae alone, respectively, while, the elements existed in the-665 to-1,475 bp fragment had enhanced effect on Verticillium dahliae. The-1,475 to-1,890bp fragment contained some negative regulated elements. Based on this transgenic analysis of GbRLK promoter, we concluded that the GbRLK plays a key role on regulation the response to stress in plant.Furthermore, we transformed the over-expression vector derived by the constitutive CaMV35S promoter in pBI121 into the cotton WO genome by the Agrobacterium-mediated transformation and obtained four homozygous transgenic lines. Based on the transgenic cotton phenotypic results of resistance, we found the resistence of the transgenic cotton to Verticillium dahlia was the same as the non-transgenic receptor WO at earlier stage of inoculation, but they showed significant differences at later stage of inoculation and transgenic cotton presented higher resistance to Verticillium dahliae. Taking the pathogenicity of V. dahliae on cotton into account, we supposed that resistance to Verticillium dahliae in transgenic GbRLK cotton/Arabidopsis might result from reducing the damage of water deficiency.