Identification Of TCP Gene Family And The Initial Establishment Of CRISPR-Cas9 Gene Editing System In Brassica Rapa Chinensis

Brassica rapa L.ssp.chinensis(var.utilis Tsen et Lee)belongs to the cruciferous Brassica breeds.It is one of characteristic vegetables in southern China,which occupies an important position in the daily supply of vegetables.The leaves are the main edible parts of this vegetable,so the leaf type directly determines the quality and the value.The TCP transcription factor family have been proved to play important rolesin the regulation of plant growth,flower development and other important life processes like leaf morphogenesis,therefore the study of TCP gene family functionsin Brassica species will greatly facilitate the understanding of leaf development and morphologyfor the selection of valuable leafphenotypes of new Brassica varieties for commercialapplication.Traditional breeding methods have many limitations,selectively,the CRISPR-Cas9 gene editing system provides an effective way to conquer these drawbacks.This system uses a specific small guide RNA(sgRNA)and a CRISPR-Cas RNA-guided nuclease(Cas9)to result in gene modification,insertion or knockout,which is the most efficient and convenient gene editing system.Recent years,the application of this system was widely applied in many kind of plants and animal species.However,the report of the application of this system in Brassica species is rarely.In this study,by using bioinformatics tools,39 Brassica rapachinensis genome encodedTCP genes were identified.Their homology and collinearity relationships with the TCP gene family of Arabidopsis werecompared.After that,the Brassica rapacotyledon petiole induced tissue regeneration system was optimized and the genetic transformation system byusing this optimized regenerationsystem to transformation of the CRISPR-Cas9-BrCP14-sgRNA was preliminaryexploration.These work providea foundation for the establishment of CRISPR-Cas9 system in Brassica rapa.The main results are listed as follows:1.Identification of Brassica rapa chinensis TCP gene family.The CDS sequences of all 39 members of the TCP gene family in Brassica rapa genome were obtained from the BRAD website.The full length sequences of the TCPs in the whole genome of Brassica rapa and Arabidopsis thaliana were sorted by Phytozome and BRAD,and theirhomology was analyzed by using the neighbor-Joining method of MEGA software to further understanding the evolutionary relationship of TCP genes between the two species.The above genes were mapped to the corresponding chromosome locusby using the free visualization software Circos and their collinearity was predicted.The semi-quantitative PCR primers were designedby the online primer designingsoftwareQuantPrime.The tissues from 3-week-old plantlets were used for expression analysisof Brassica rapa TCP genes.Roots,the stage ?,? and ? apical meristem,the cotyledons,first and second leaf,the hypocotyls and petioles were collected and usedfor total RNA isolated.The total RNA from each tissue was reverse transcribed into cDNA and the transcription level of each member of the 39 TCP transcription factor genes was detected by semi-quantitative PCR.It was found that total 25 genes show expression in the detected tissues,and overall show higher expressionlevel in the second leaf and petiole than in the other tissues.Eight TCP genes show complete expression in all the tissues,whereas others show expression specificity in different tissues,which indicate their important and diverse functions in regulating leaf morphogenesis during the leaf development progress.2.Establishment of Brassicarapa cotyledon petiole callus induction and tissue regeneration system and the preliminary exploration of Genetic transformation system system in this species.The effects of different concentrations of 6-BA,NAA,gelzan,AgN03 and other factors on the induction and regeneration of Brassicarapa cotyledon petiole callus were investigated by using seeds as the initial material.The culture medium containing lx MS(M519)+ 2mg/L 6-BA + 0.45mg/L NAA + 4mg/L AgN03 + 20 g/L sucrose + 3 g/L gelzan(pH 5.8)was detected andconsidered to be the best for cotyledon petiole callus induction and regeneration.The transformation of cotyledon petioleexplants was applied by Agrobacterium tumefaciens mediated method.Through explorations of the antibody resistance sensitivity,the types and concentration of various bacteriostatic agents,the recovery culture and other factors,the genetic transformation system suitable for Brassica rapa was preliminary established.The optimal screening concentration of hygromycin is the 6mg/L,the best antibacterial agent is carbenicillin and its optimal concentration is 200mg/L.After co-culture,the explants were difficult to produce callus and adventitious buds if directly transferred into differentiation culture medium,but can be successfully induced callus if recover for four days inrecovery medium and then transfer into the differentiation culture medium,but still cannot obtain transgenic adventitious buds.We suppose that hygromycinsuppresses Brassica rapa callus regeneration or the Agrobacterium tumefaciens GV3101 may not also be suitable for transformation of Brassica rapa,the change of antibiotics to kalamycin system and Agrobacteriumstain LBA4404 should solve these problem.3.According to the previous study and the results of the TCP gene family in this study,the selection of cabbage TCP14 as the target of gene editing.The vector pCBIM-Cas9-TCP14-sgRNAI and pCBIM-Cas9-TCP14-sgRNA2 were constructed by gene editing of TCP 14 by designing the sgRNA of the Chinese cabbage TCP 14 gene via the CRISPR/Cas9 online guidance RNA design website.The CRISPR/Cas9 gene editing system was explored by using the cabbage regeneration system and the genetic transformation system.Taken together,in this study,the TCP transcription factor genes of Brassica rapachinensis were identified,and the optimization of callus induction and adventitious bud regeneration system was established.Based on this system,the Agrobacterium tumefaciens-mediated CRISPR-Cas9 gene editing and genetic transformation system of Brassica rapaspecies were preliminary explored.These workprovide valuableknowledge of the TCP transcription factor gene family in Brassica rapa,their evolutionary relationship with the ArabidopsisTCP gene family,and their possible relations with leaf development and morphogenesis.The callus induction and adventitious bud regeneration system would provide useful tool for studying gene functions in Brassica rapa species in future,and the utility of the CRISPR-Cas9 gene editing system will greatly accelerate the speed of new germplasm innovation ofBrassica species like various leaf morphology in future.