Study On The Function Of CpYABBY5-1 And CpCRC Of YABBY Family In Chimonanthus Praecox

Chimonanthus praecox,a perennial deciduous shrub,is an important traditional ornamental flower and winter fragrant flower plant in China.In this study,five members of CpYABBY family including CpYABBY5-1,CpYABBY5-2,CpYABBY2,CpFIL and CpCRC were identified by using the whole genome of C.praecox,and the functions of CpYABBY5-1 and CpCRC were studied emphatically.The main results are as follows:Five members of the YABBY family of C.praecox were identified as Cp YABBY5-1,Cp YABBY52,CpYABBY2,CpFIL and CpCRC.Among the family members,the shortest coding sequence of CpCRC was only 527bp,and the longest coding sequence of CpFIL was 646bp.Except for CpYABBY5-1 and CpFIL,CpYABBY5-2,CpYABBY2 and CpCRC all contained 6 introns.By analyzing the proteins encoded by family members,it was found that all members were hydrophilic proteins and subcellular localization predictions were located in the nucleus.Sequence alignment of family members showed that CpYABBY contained two conserved domains:a C2C2 zinc finger domain(zinc finger-like)at the N-terminal and a YABBY(Helix-loop-helix)domain at the C-terminal.The prediction results of the promoter region of CpYABBY gene showed that there were 16 cis-acting elements involved in growth and development,hormone response and stress response of CpYABBY family genes.The results of phylogenetic relationship of YABBY family showed that CpYABBY could be divided into five categories:INO Clade,CRC Clade,FIL Clade,YABBY5 Clade and YABBY2 Clade of YABBY family.The species with close relationship to C.praecox were C.salicifolius,C.micranthum and V.yinifera.CpYABBY5-1 and CpCRC from the CpYABBY family were selected as key research genes,and their expression characteristics in C.praecox were analyzed by qRT-PCR technique.It was found that CpYABBY5-1 gene was expressed in roots,leaves,flowers and young fruits of C.praecox,with the highest expression in the root,the second in the leaf and the lowest in the stem.In terms of time distribution,the maximum expression of CpYABBY5-1 gene was during branch and bud germination in early March.While CpCRC gene was expressed in leaves,stamens,pistils and young fruits of C.praecox,with the highest expression in pistils and the lowest in leaves.In terms of time distribution,the expression of CpCRC gene fluctuated with the stage of flower bud differentiation.C.praecox seedlings with consistent growth were treated with drought,high salt and low temperature.The results showed that under high salt and low temperature stress,the expression of Cp YABBY5-1 gene increased at first and then decreased,while under drought stress,the expression of CpYABBY5-1 increased at first and then decreased.Different expression patterns may indicate that CpYABBY5-1 gene plays different functions and roles in seedlings under different abiotic stress.In this research,the overexpression vectors of CpYABBY5-1 and CpCRC were successfully obtained.The RNA of T2 generation transgenic A.thaliana was extracted and the expression of each line was determined by qRT-PCR.Finally,it was determined that OE10-5 and OE8-6 lines represented 35S::CpYABBY5-1/Col-0,while 35S::CpCRC/Col-0 were represented by OE6-10 and OE8-12 lines.The phenotypes of the above lines were observed,and follow-up experiments were carried out.In the process of studying the function of CpCRC gene,we introduced crc-1 A.thaliana seeds,sowed them with the seeds of WT,OE6-10 and OE8-12,and cultured them routinely.Meanwhile observation and comparison showed that the crc-1 mutant line completed vegetative growth early and quickly transferred to reproductive growth,showing a growth trend of early bolting,early flowering and early fruiting,while heterologous expression of CpCRC may accelerate the growth and development of A.thaliana to some extent.Before bolting 1cm,the growth rate of OE5-10 and OE812 was significantly slower than that of WT,but at the time of the first flowering and the appearance of the first pod,35S::CpCRC/Col-0 caught up,which may indicate that CpCRC gene began to express or play a role after bolting 1cm in A.thaliana.The leaf type of WT and crc-1 lines showed oval or narrow oval style,while the heterologous expression of CpCRC caused the stem leaf of A.thaliana to curl to the main axis of leaf vein as a whole,resulting in a sharp decrease in leaf width and a sharp increase in leaf shape index.There was no significant difference in the number and size of lateral nectary between WT and 35S::CpCRC/Col-0.The shape of lateral nectary of the latter was relatively slender,while that of crc-1 disappeared completely.In addition,after observing the pod development of 35S::CpCRC/Col-0,WT and crc-1 lines,it was found that the pods of OE5-10 and OE8-12 were thinner and longer,the number and arrangement density of seeds were higher than those of WT lines.On the other hand,the pods of crc-1 line were not fully developed,which showed that the carpels at the top of the pods were not fused,showing a "concave" type,and the internal seeds were loosely arranged and the number decreased significantly,which indicated that CpCRC gene may play a certain role in the mechanism of ovule formation in A.thaliana.In the process of studying the function of CpYABBY5-1 gene,the conventional cultured WT,OE10-5 and OE8-6 were observed and treated under drought condition,NaCI simulated high salt condition and low temperature stress respectively,and multiple physiological activity indexes of A.thaliana were measured.Data analysis showed that heterologous expression of CpYABBY5-1 not only enhanced A.thaliana tolerance to high salt and low temperature stress,but also more sensitive to drought stress.The leaves of different lines were collected after treatment,and the accumulation degree of H2O2 and O2-in leaves was shown by DAB and NBT methods,and the staining results were consistent with the results of physiological indexes.When discussing the cold tolerance of 35S::CpYABBY5-1/Col-0,the modulated chlorophyll fluorescence imaging system(Walz,Germany)was used to photograph and measure the chlorophyll fluorescence parameters of OE10-5,OE8-6 and WT lines after low temperature treatment.The parameters of Fv/Fm,NPQ_Lss and Rfd_Lss;confirmed that the heterologous expression of CpYABBY5-1 gene enhanced the tolerance of A.thaliana to low temperature.Under NPQ_Lss parameters,the tolerance of transgenic A.thaliana to low temperature was positively correlated with the expression of CpYABBY5-1 gene.The leaves of WT,OE10-5 and OE86 were collected and placed in simulated drought,high salt and low temperature,respectively,and then the stomatal conductance of the back of the microscope was changed,at the same time results once again verified the above conclusion.To sum up,CpCRC plays a decisive role in determining leaf abaxial polarity.Heterologous expression of this gene will lead to serious curl of A.thaliana cauline leaves and decrease of leaf width and length.Compared with crc-1 and WT lines,CpCRC gene may promote the development of A.thaliana pistil carpels.In addition,CRC is a necessary gene in the development of lateral nectaries in plants,which plays a unique role in pistil carpel fusion and ovule development.CpYABBY5-1 gene is a positive regulator of salt and low temperature stress response and a potential gene to improve salt and low temperature resistance of C.praecox.Heterologous expression of this gene will enhance A.thaliana tolerance to salt and low temperature stress,but it will also make it more sensitive to drought.The functional exploration of the two YABBY genes reflects the multifaceted gene function of the family,which not only provides genetic resources for improving the stress resistance and flower aroma of C.praecox,but also provides a new way to further explore the genetic background of C.praecox.