| Natural rubber biosynthesis is a typical plant secondary metabolic pathway,which is closely related to plant hormone signal transduction,wounding and other stress,and its synthesis is also regulated by stress factors such as light and pests.The MYB protein family is a large and diverse function family that plays an important role in plant growth and development,stress resistance,and secondary metabolism.However,the mechanism by which the MYB transcription factor family members transcriptionally regulate the natural rubber biosynthesis is still unclear.Taraxacum kok-saghyz Rodin is an ideal model system for studying the biosynthesis of natural rubber.Based on previous studies that HbMYB44 interacts with MBS motif in the promoter region of genes related to natural rubber biosynthesis,in order to systematically analyze the regulatory mechanism of MYB family member HbMYB44 in natural rubber biosynthesis,molecular biology and genetic transformation techniques were used to obtain transgenic Arabidopsis and transgenic Taraxacum kok-saghyz Rodin plants and performed functional verification.The results were as follows:1.We first cloned the full-length c DNA of HbMYB88 from the leave of rubber tree Reyan73397.It is 1848 bp in length,containing a 1440 bp ORF which encodes479 amino acid residues.The deduced amino acid sequence of HbMYB88 contains two SANT conserved domains,has HTH tertiary structure,share highly similarity to Arabidopsis At MYB88,At MYB124.The subcellular localization results indicated that HbMYB88 was localized in the nucleus.By q RT-PCR analysis,HbMYB88 was mainly expressed in the stem and flower of rubber tree,whilst its expressions in root,leaf,bark and latex were extremely low.The expression level of HbMYB88 in tissue culture seedling leave was significantly upregulated under the treatment of hydrogen peroxide(H2O2),and abscisic acid(ABA).These indicated that HbMYB88 has a relationship with stress response such as drought in rubber trees.2.Bioinformatics analysis showed that HbMYB44 has two conserved domains and clustered into the S22 subfamily,which is related to stress resistance.q RT-PCR results showed that HbMYB44 is responsed to various treatments such as mechanical wounding,drought,ABA,ETH,Me JA,SA and so on.It is suspects that HbMYB44 is the key in multiple signal pathway.3.The HbMYB44 overexpression vector was constructed and obtained Arabidopsis thaliana T3 generation by Basta selection.Compared with WT,HbMYB44-OE Arabidopsis lines increased the response to drought,salt resistance and gibberellin under hormones and stresses treatment.These suggested that overexpression of HbMYB44 in Arabidopsis increased the stress resistance function of transgenic Arabidopsis.4.Positive HbMYB44-OE transgenic plants were obtained through genetic transformation technology.The q RT-PCR analysis confirmed that the transcript abundance of many natural rubber biosynthesis-related genes(Tk SRPP,Tk REF,and Tk CPT)was upregulated in Taraxacum kok-saghyz Rodin OE lines and WT 181-4lines.Transcriptome data showed that overexpression of HbMYB44 in Taraxacum kok-saghyz Rodin increased the expression of MYB family S22 members(Tk MYB44,Tk MYB73,Tk MYB77),ABA receptor genes(Tk PYL8,Tk PYL9,Tk RAB18),and natural rubber biosynthetic genes(Tk SRPP,Tk CPT).It is confirmed that overexpression of HbMYB44 in Taraxacum kok-saghyz Rodin significantly increased the natural rubber synthesis via enhanced their expression.5.We proved that HbMYB44 interacted with HbSRPP1 in yeast and tobacco by the yeast two-hybrid and Bimolecular Fluorescent Complimentary respectively.Firefly luciferase complementary imaging experiments show that HbMYB44 interacted with HbSRPP1.These indicated that HbMYB44 interacted with HbSRPP1 to regulate natural rubber biosynthesis.6.To sum up,a model of HbMYB44 regulating natural rubber biosynthesis and stress resistance is proposed,which lays the foundation for elucidating the molecular mechanism of HbMYB44 transcription regulating natural rubber biosynthesis. |
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