Cloning And Functional Verification Of Anthocyanin Related Gene BsRbohD In Begonia Semperflorens

Begonia semperflorens is a kind of perennial herbaceous flower,which is often used as parterre and ground cover.Among them,the widely used green leaf Begonia ‘super Olympics’ varieties often turn red because of the accumulation of anthocyanins due to environmental changes.Anthocyanins are the main substances that make the plant tissues appear red to purple,so they are known as "indicators" of plant stress.Therefore,B.semperflorens can be used as an excellent research material in the study of environmental induced anthocyanin synthesis.In order to explore whether the accumulation of anthocyanin synthesis in B.semperflorens leaves under low temperature and high light environment is directly related to the production of reactive oxygen species mediated by NADPH oxidase,this study carried out cloning analysis,tissue-specific expression analysis and subcellular localization analysis of NADPH oxidase family gene BsRbohD,which was obtained by our research group in the early stage The transgenic wild-type Arabidopsis thaliana was transformed into"Columbia" wild-type A.thaliana,and the non transgenic wild-type A.thaliana was treated with low temperature and high light respectively,and the photosynthetic characteristics,physiological indexes and expression levels of related structural genes were detected.The results were as follows:1.The NADPH oxidase family gene BsRbohD(accession number:MH784503)was successfully cloned from B.semperflorens.The total length of the gene was 3796bp,and the length of CDS region was 2784bp,encoding 957 amino acids.2.The cDNA obtained from total RNA reverse transcription of B.semperflorens leaves,male flowers,female flowers,stems and roots as templates,The results of real-time PCR showed that BsRbohD gene was expressed in leaves,male flowers,female flowers,stems and roots in B.semperflorens,but the expression levels were different in different tissues,among which root>male flower>female flower>leaf>stem.3.GFP expression vector of B.semperflorens BsRbohD gene was successfully constructed.Compared with the expression vector of marker protein of RFP plasma membrane,it was transiently expressed in tobacco leaves by injection into the lower epidermis of tobacco leaves.The NADPH oxidase protein controlled by BsRbohD gene of B.semperflorens was located on the cytoplasmic membrane by laser confocal microscope.4.The BsRbohD gene was heterologous expressed in A.thaliana,and the T3 generation positive plants were identified and screened by hygromycin HYb and genome-specific PCR amplification.Two transgenic positive lines were selected,and wild type A.thaliana was used as control.By measuring the photosynthetic fluorescence parameters of transgenic plants and wild-type plants,combined with the rapid chlorophyll fluorescence induction kinetics curve and The "JIP-test" parameter analysis showed that there were significant differences between wild-type A.thaliana and the two transgenic lines at J,I and P points,which were larger than those of the transgenic plants and there was no significant difference between the two transgenic lines.The Fv/Fm,Fv/fo,ABS/CSo,TRo/CSo,ETo/CSo,REo/CSo,Sm,ETo/RC,REo/RC,PI abs and PI total values of the two transgenic lines were lower than those of wild-type A.thaliana,while the values of Fo/Fm,Vj,Vi,N,ABS/RC,DIo/RC,TRo/RC and DIo/CSo values of the two transgenic lines increased,indicating that the heterologous expression of BsRbohD gene in A.thaliana changed the physiological state and photosynthetic mechanism of leaves of A.thaliana.After stress,the physiological indexes and relative expression of related genes were measured,and the results showed that the transgenic lines were more sensitive to environmental mutations than wild-type plants.The expression of superoxide anion production rate,hydrogen peroxide content,anthocyanin content and BsRbohD gene were up-regulated faster than wild-type A.thaliana,and the content or expression of BsRbohD gene was higher than that of wild-type A.thaliana.The expression levels of several anthocyanin biosynthesis genes in A.thaliana were higher than those in wild-type plants at the late stage of stress,among which the sensitivity to high light stress was greater than that of low temperature stre ss,which indicated that the BsRbohD gene of B.semperflorens cloned in this research could express normally in A.thaliana,and its NADPH oxidase function was normal,and it could produce reactive oxygen species to cope with environmental changes.The reactive oxygen species produced by BsRbohD gene can be used as signal molecules to directly or indirectly regulate the synthesis and accumulation of anthocyanins.