| About 10%arable land(total 800 million hm~2)is affected by salinization worldwide,accounting for 4.88%arable land(or 3.6×10~7 hm~2)in China.However,with growth of the world population,global food supply have faced great threaten and soil salinization is still increasing.Therefore,how to fundamentally solve the soil salinization problem have attracted much attention.Halophytes can resist high salt conditions and complete their life cycle under Na Cl treatment.So they provide feasible models to study salt resistance and improve crop salinity tolerance using transgenic techniques.Limonium bicolor is a typical recretohalophyte with salt glands on its leaf surface,which is considered a model halophyte to study salt gland development.Previous studies have systematically elucidated the salt glands morphological structure,but fewer studies focused on its in-depth salt secretion and developmental mechanism.Given that LbTRY was involved in salt glands development based on the previous study,this thesis deeply discusses the role and molecular mechanism of LbTRY in salt gland development,which provides auxiliary data for the future study of salt gland development.The main experimental results are as follows:1.LbTRY gene negatively regulates the salt gland development and salt resistance in L.bicolorUsing CRISPR-Cas9 gene editing technology and overexpression technology,knockout lines(LbTRY-CR)and overexpression lines(LbTRY-OE)were obtained.According to mature salt gland generated obviously bright blue autofluorescence under UV excitation,the phenotypic of regenerated buds were observed.Compared with the salt glands of wild-type with four autofluorescence foci,LbTRY-CR salt glands had more autofluorescence foci,and abnormal rate was 45.64%,while LbTRY-OE salt glands had fewer autofluorescence foci,and abnormal rate was 50.3%.These results indicated that LbTRY overexpression inhibited salt glands differentiation.VIGS technology was used to silence the six-leaf stage L.bicolor,and observed salt gland development and salt secretion of silent lines.After silencing LbTRY,the salt secretion increased significantly,the salt glands total number also increased under DIC microscope observation.Some salt glands had more autofluorescence foci.DAB and NBT staining became shallow,indicating the silencing strain of LbTRY gene significantly improved the stress resistance of plants.Based on the above results,LbTRY gene is involved in salt glands development and salt resistance as a negative role.2.LbTRY interaction protein screening and verification of L.bicolorLbTRY interaction proteins were screened by nuclear yeast two-hybridization technology.According to the evidence of homology and high expression of candidate genes during salt glands development,the candidate interaction proteins of LbTRY were obtained.Lb3G20842and Lb7G34824 were finally screened by yeast two hybrid verification and bimolecular fluorescence complementary experiments.Two interacting proteins also had an interaction relationship.The mechanisms of LbTRY gene and interaction protein involved in salt glands development were further explored.3.Lb3G20842 gene and Lb7G34824 gene are localized in nucleus and specifically expressed in epidermal structureThe length of Lb3G20842 was 846 bp,encoding 281 amino acids.Its gene expression pattern analysis showed high expression in the early stage of salt gland development,and subcellular localization was on the nucleus.Its promoter is cloned 2000 bp,and the element analysis contained salicylic acid reaction element(TCA-element)and other reaction elements.GUS expression vector containing Lb3G20842 promoter,pCAMBIA3301-pLb3G20842-GUS,was transformed in Arabidopsis.Lb3G20842 gene expressed in cotyledon vein,the first true leaf trichomes,growth point,rhizome junction and taproot.This result indicated that Lb3G20842gene regulated epidermal structure development.The length of Lb7G34824 was 657 bp,encoding 218 amino acids.Its gene expression pattern analysis showed high expression in the early stage of salt gland development,and subcellular localization was on the nucleus.Its promoter is cloned 2000 bp,and the element analys contained methyl jasmonate reaction elements(TGACG-motif,CGTCA-motif).GUS expression vector containing Lb7G34824 promoter,pCAMBIA3301-pLb7G34824-GUS,was transformed in Arabidopsis.Lb7G34824 gene expressed in cotyledons,the first true leaf trichomes,growth point,rhizome junction,primary root and lateral root.The result indicated that Lb7G34824 gene regulated epidermal structure development.4.The mechanism investigation of Lb3G20842 gene and Lb7G34824 gene are involved in salt glands development of L.bicolorIn order to investigate the functions of Lb3G20842 and Lb7G34824,VIGS techonology was used to silence the six-leaf stage L.bicolor,and observed salt gland development and salt secretion of silent lines for the purpose of exploring mechanisms of Lb3G20842 gene and Lb7G34824 gene involved in salt gland development.After silencing Lb3G20842 gene and Lb7G34824 gene,the salt secretion decreased significantly,the salt glands total number also decreased under DIC microscope observation.DAB and NBT staining became deeper,indicating silencing Lb3G20842 gene and Lb7G34824 gene decreased the salt stress resistance of plants.Therefore,Lb3G20842 gene and Lb7G34824 gene are involved in salt glands development as positive roles.Using CRISPR-Cas9 gene editing technology and overexpression technology,knockout lines(Lb3G20842-CR)and overexpression lines(Lb3G20842-OE)were obtained.According to mature salt gland generated obviously bright blue autofluorescence under UV excitation,the phenotypic of regenerated buds were observed.Compared with the salt glands of wild-type with four autofluorescence foci,Lb3G20842-CR salt glands had fewer autofluorescence foci,and abnormal rate was 42.5%.These results indicated that Lb3G20842 knockout inhibited salt glands differentiation.Using CRISPR-Cas9 gene editing technology and overexpression technology,knockout lines(Lb7G34824-CR)and overexpression lines(Lb7G34824-OE)were obtained.According to mature salt gland generated obviously bright blue autofluorescence under UV excitation,the phenotypic of regenerated buds were observed.Compared with the salt glands of wild-type with four autofluorescence foci,Lb7G34824-OE salt glands had more autofluorescence foci,and abnormal rate was 33.47%.These results indicated that Lb7G34824 overexpression increased salt glands differentiation.5.LbTRY,Lb3G20842 and Lb7G34824 co-regulatied salt glands developmentThe co-regulation of LbTRY,Lb3G20842 and Lb7G34824 was comprehensive analyzed based on the silencing phenotypes in L.bicolor silent of LbTRY and two interacting protein genes(Lb3G20842 and Lb7G34824).LbTRY negatively regulated salt gland development and salt resistance,while two interacting proteins positively regulated salt gland development and salt resistance.The expression levels of Lb3G20842 and Lb7G34824 in LbTRY silencing lines were significantly upregulated by q RT-PCR,while in Lb3G20842 or Lb7G34824 silent lines,the expression level of LbTRY showed a downward trend.It was indicated that LbTRY,Lb3G20842and Lb7G34824 genes formed a negative regulatory complex dominated by LbTRY gene in regulating the salt glands development.LbTRY regulates the expression of Lb3G20842 and Lb7G34824 by negative feedback regulation,thereby controlling the salt glands development.The specific interaction mechanism and regulation mode need to be further studied. |
PDF Full Text Request