| Oxalis triangularis subsp.papilionacea is often planted as a horticultural plant in various potted plants and gardens because of its unique purple appearance.Anthocyanins,as a kind of water-soluble flavonoid pigments,can not only make plants present colorful colors from yellow to blue,but also play a role in plant stress resistance.At present,the mechanism of anthocyanin biosynthesis and molecular regulation has been clearly described in many plants.However,the mechanism of anthocyanin biosynthesis in Oxalis is still blank,and even the genome sequence of Oxalis has not been reported still.In this study,Oxalis triangularis subsp.papilionacea and Oxalis corymbosa DC were used as experimental materials,and their leaves were transcriptome sequenced to explore the molecular mechanism of anthocyanin synthesis.The main results are as follows:（1）Phenotypic analysis and anthocyanin content analysis showed that leaves of Oxalis triangularis subsp.papilionacea are purple and flowers of them are lilac,while leaves of Oxalis corymbosa DC are green and flowers of them is purplish red.The anthocyanin content of leaves in Oxalis triangularis subsp.papilionacea are much higher than that of Oxalis corymbosa DC,while the anthocyanin content of Oxalis triangularis subsp.papilionacea flowers is lower than that of Oxalis corymbosa DC.The results of LC-MS/MS showed that there are three kinds of anthocyanins in leaves of Oxalis:Malvidin3-O-（4-O-（6-O-malonyl-glucopyranoside）-rhamnopyranosyl）-5-O-（6-O-malonyl-glucopyranoside）,Malvidin-3,5-di-O-glucoside（Malvin）,Delphinidin-3-O-rutinoside,and the fold change values between the two kinds of materials are 476666.67,25.93 and 36.06,respectively.（2）The genes related to anthocyanin in leaves of Oxalis triangularis subsp.papilionacea and Oxalis corymbosa DC were screened,and seven pathway genes involved in anthocyanin biosynthesis were identified:PAL,CHS,CHI,F3H,F3’5’H,ANS and UA3GT,all of which were up-regulated in leaves of Oxalis triangularis subsp.papilionacea;and six transcription factors genes related to anthocyanin regulation were also identified:MYB113,TT8,TTG1,GL3,TTG2,CPC,in which the expression of MYB113 and TT8 were up-regulated in the leaves,while the expression of TTG1,GL3,TTG2 and CPC were down-regulated in the leaves.（3）According to the results of transcriptome sequencing,the anthocyanin related genes in leaves and flowers of Oxalis triangularis subsp.papilionacea and Oxalis corymbosa DC were detected by q RT-PCR.The results showed that the expression levels of pathway genes PAL,CHS,CHI,F3H,F3’5’H,ANS,UA3GT and transcription factors MYB113,TT8 were up-regulated in leaves of Oxalis triangularis subsp.papilionacea,and the pathway genes F3H,F3’5’H and transcription factor TTG2 were down-regulated in flowers of Oxalis triangularis subsp.papilionacea,which indicated that pathway genes PAL,CHS,CHI,F3H,F3’5’H,ANS,UA3GT and transcription factors genes MYB113,TT8 might be the genes promoting anthocyanin accumulation in leaves of Oxalis triangularis subsp.papilionacea,while F3H,F3’5’H and TTG2 might be the genes causing anthocyanin content up-regulation in flowers of Oxalis corymbosa DC.（4）The seedlings of Oxalis triangularis subsp.papilionacea were treated in low temperature and dark condition.The results showed that the anthocyanin accumulation was promoted to a certain extent under low temperature and was greatly inhibited under dark condition.The q RT-PCR results of related genes showed that the promotion of anthocyanin synthesis under low temperature might be caused by the increased expression of CHS,CHI and transcription factors genes TT8,TTG1,GL3 and TTG2,while the inhibition of anthocyanin synthesis under dark condition might be caused by the decreased expression of pathway genes F3H,F3’5H and transcription factors MYB113,TT8.In addition,a small amount of anthocyanins still existed in Oxalis triangularis subsp.papilionacea in dark conditon,which indicated that there might be another pathway for anthocyanin synthesis of Oxalis triangularis subsp.papilionacea not limited by light.（5）Soluble sugar,soluble protein and total phenol in Oxalis triangularis subsp.papilionacea leaves under low temperature and dark stress were detected.The results showed that soluble protein and soluble sugar could promote anthocyanin synthesis of Oxalis triangularis subsp.papilionacea leaves under low temperature and dark stress.Low temperature promoted phenol synthesis,while dark inhibited phenol production.（6）The transcription factor Ot MYB113,which is the most up-regulated transcription factor in transcriptome sequencing,is cloned to construct its over-expression vector p BI121-Ot MYB113 and transformed into cotyledons of wild type tomatoes AC++to induce formation of callus.However,there was no significant change in the callus,suggesting that Ot MYB113 may not be the key gene for the formation of purple leaves on Oxalis triangularis subsp.papilionacea.On the other hand,sequence analysis showed that the stop codon of Ot MYB113 appeared at 270 bp from the start codon of it,which made the Ot MYB113 only contain 89 amino acids.Compared with other species of MYB transcription factors,the Ot MYB113 sequence cloned in this study only contains DNA binding domain and lacks transcriptional activation domain,which made it unable to increase the content of anthocyanins in Oxalis triangularis subsp.papilionacea,indicating that there are other MYB transcription factors related to anthocyanin synthesis promotion that we have not found in Oxalis triangularis subsp.papilionacea.In conclusion,it is anthocyanin that affects the leaf color and flower color of Oxalis.The anthocyanin synthesis pathway genes PAL,CHS,CHI,F3H,F3’5H,ANS,UASGT and transcription factors MYB113 and TT8 are related to anthocyanin accumulation in Oxalis leaves.The pathway genes F3H,F3’5’H and transcription factor gene TTG2 were related to anthocyanin accumulation in flowers of Oxalis corymbosa DC.Besides,low temperature promoted anthocyanin accumulation in Oxalis corymbosa DC to a certain extent,while it can be inhibited in dark condition. |
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