Cloning And Functional Analysis Of BoHB12 And BoHB7 Genes Associated With Leaf Curling In Cabbage (Brassica Oleracea Var. Capitata L.)

Cabbage (Brassica oleracea var. capitata L.) is a member of genus Brassica of Cruciferae plant, which is one of the most important leaf vegetable in the world, and plays an important role in the development of vegetables in China.In the period of vegetation, cabbage experiences seedling stage, rosette stage, and heading stage. The leaf grow inward and upward, then become curl in the heading stage. Curly leaves not only break the leaf flatness, but also influence physiology in most plants, such as photosynthesis, transpiration and respiration. However, it is very helpful for cabbage whose eating position is leaf head. The number and the degree of curl leaves affect the weight and firmness of head directly, which are two commodity traits of cabbage. Formation of head is a complicated biological process, which is influenced by multiple environmental factors, such as temperature, light, nutrition, and endogenous homone. Leaf curl inward is an important trait to reflect the formation of head. And cabbage with curly leaves is an ideal material to analyze the mechanism of leaf curl. Cause gene expression is key regulation when plant gets the signal from the environment, focusing on expression of these genes associate with leaf curl would help to make sense the mechanism of head formation in cabbage and produce varity with good quality and high yield cabbage via molecular strategy.Homeobox (HB) protein is a kind of important transcription factors related to plant development. HB gene family have highly similar conserved protein domain, called homeodomain(HD), consisting of sixty or sixty-one amino acids. The plant homeoboxes gene family is classified into six subfamilies based on the paralog of conserved amino acids sequence and the sequence on both sides of HD. In which, HD-Zip is a kind of specific transcription factors in higher plants and is subdivided into four classes:Ⅰ,Ⅱ, Ⅲ,Ⅳ. HD-Zip plays an important role to regulate plant cell differentiation. In the previous study, leaf polarity regulatory gene HYPONATIC LEAVES1 regulated gene expression of class Ⅲ from HD-Zip by mediating miRNA in Arabidopsis thaliana. In the mutant with hyl1 knocked down of Arabidopsis thaliana, leaves curled inward and PHB, PHV and REV genes over-expressed. It reflected that HD-Zip gene family might regulate the leaf curled.In the present study, cabbage’519’was used to detect the function of HB gene in regulating leaf curvature during formation of head in cabbage. HB genes were analyzed by transcriptome sequencing. HB genes associated with curly leaves were cloned via comparing leaf, head morphology and tissue structure at rosette stage. These genes’ function was identified by HB genes transformation into cabbage and analyzing GUS gene expression at the promoter of target gene. It indicated that HB genes played the important role to regulated leaves curvature during heading in cabbage.The main results are as follows:1. Comparison leaves and cell tissue structure at the rosette stage with heading stage.We observed the phenotype of leaves at the rosette stage and head at the heading stage, the cytology of leaves from different position by paraffin method. The head leaves lack of petiole was light yellow, curled upward, smaller leaf index, while the rosette leaves were green flat with petiole. Compared with the rosette leaves, the head leaves were thicker, delayed mesophyll cell differentiation. Only mesophyll cell from the top of leaves at heading stage, differentiate into palisade parenchyma and spongy parenchyma, while it finished differentiation in the middle of leaves at rosette stage.The phenotype of head leaves was observed under high temperature stress and pressing leaves treatment. The 34th leaf of head leaf was curly without stress, but was similar with rosette leaf after treatment. It indicated that high temperature and light might affected leaves heading and leaves curling of cabbage.2. Cloning and expression analysis of homeobox genes from cabbage selected by the transcriptome analysis.Four significantly differentially expressed HB paralogous genes, BoHAT2, BoHB12, BoHB7, BoHB27 were identified via transcriptome comparison of stem tip and leaf at rosette stage and heading stage of cabbage ’519’.BoHAT2, BoHB12, BoHB7, BoHB27 were cloned together with other three paralogous HB genes BoPHB, BoPHV, BoREV. These genes were not identified with different gene expression at rosette stage and heading stage of cabbage by transcriptom sequencing, but regulate leaf curl in Arabidopsis. All the seven genes shared a homeodomain of homebox family according to amino acids sequence. They were highly homologous with HB protein family from Arabidopsis thaliana via BlastP analysis. The similarity of amino acids sequence of BoHB12, BoHB7, BoHAT2, BoHB27, BoPHV, BoPHB, BoREV genes are 80%,79%, 86%,60%,95%,94%,96% compared with ATHB12, ATHB-7, HAT2, ATHB27, PHV, PHB, REV from Arabidopsis thaliana, respectively. BoHAT2 was classified into HD-Zip Ⅱ protein, BoHB12 and BoHB7 were members of HD-Zip Ⅰ protein, BoHB27 was a member of ZF-HD protein, BoPHB, BoPHV and BoREV were classified into HD-Zip Ⅲ protein according to the results of BlastP and phylogenetic tree. BoHAT2, BoHB12, BoHB7, BoHB27, BoPHB, BoPHV and BoREV genes were expressed in stem tip and leaf from rosette stage to heading stage. The BoHB12 and BoHB7 genes were over-expressed in the leaf of heading stage than the leaf of rasette stage, they were 38.1 times and 6.2 times, respectively. Others were similar gene expression. It demonstrated the BoHB12 and BoHB7 genes might be the major genes to regulate the process of cabbage leaf curl.3. Expression future of promoter from BoHB12 and BoHB7 in the transgenic tobaccopCABarBoHB12PGUS and pCABarBoHB7PGUS plant expression vectors were constructed including BoHB12 promoter-GUS and BoHB7 promoter-GUS, respectively. These two genes were transformed into tobacco by agrobacterium tumefaciens mediated transformation. Gene function of transgenic plants were identified by stained with 5-bromo-4chloro-3-indoly-β-D-glucuronide. BoHB12 promoter induced GUS expressed in leaf, stem, and root tips, and down-expressed in base and top of younger leaf. While BoHB7 promoter induced GUS expression in leaf, stem, and root, it induced GUS low-expression in younger leaf.4. BoHB12, BoHB7genes and their antisense genes transformation of cabbageTarget genes were inserted into pBI525 vector at two different sites and the direction of inserted genes can be identified by PCR amplification via using the gene’s upstream primer and NOS-R primer. The sense genes and antisense genes expression boxes were constructed at the same time. Subsequently the sense genes and antisense genes expression boxes were insert into PCAMBIA1300-bar vector to construct the plant expression vectors. Four vectors:pCABarBoHB12, pCABaranti-BoHB12, pCABarBoHB7, and pCABaranti-BoHB7 were constructed. Lastly, three genes were transformed into cabbage by Agrobacterium tumefaciens mediated transformation except that 35S::anti-BoHB7 tansgenic adventitious buds didn’t generate adventitious roots. Morphological change was oberved of 35S::anti-BoHB12 tansgenic plants. The rosette leaves of anti-BoHB12 tansgenic plants became smaller, folding and curled downward and they couldn’t shape head, which were quite different from no-transgenic plants. Expression of BoHB12, BoGA20ox1, BoGA20ox2, BoASAl, BoILL6, BoIAA12, BoTCP4, BoPHB and BoPHV in 35S::BoHB12 plants and 35S::anti-BoHB12 plants were identified by RT-qPCR. BoHB12 was depressed in 35S::BoHB12 plants rosette leaves, whereas BoHB12 was over-expressed in 35S::anti-BoHB12 plants rosette leaves. BoHB12 positively regulated BoGA20oxl expression, but negatively regulated BoIAA12 and BoPHV expression.