Evolutional Analysis And Functional Identification Of BnaA06.mTERF1 Gene In Brassica Napus L.

Rapeseed is one of the most important oil crops in China,which occupies an important position in domestic edible vegetable oil.However,the planting area and production of rapeseed has been in a state of stagnation in past decade and less than 35 percentage of consumption of edible vegetable oil in China is supplied by domestic rapeseed.In order to ensure the food security in China,the most effective way is to increase rapeseed yield.Chlorophyll is an important pigment for photosynthesis,and its content directly affects the photosynthetic capacity of plants,and thus indirectly affects the acquisition of energy and growth and development of plants.Therefore,it is of great significance to study chlorophyll to improve photosynthetic efficiency of plants and increase crop yield.In higher plants,mitochondrial transcriptional termination factor(m TERF)has biological functions such as affecting the gene expression and DNA replication of mitochondria or chloroplasts,chloroplast development,embryonic development and plant response to adversity.Bioinformatics analysis of Bna A06.m TERF1 gene was performed,and its function was identified based on functional complementary experiment with Arabidopsis mutant and CRISPR/Cas9 knockout experiment with the Zhongshuang 11 in Brassica napus.In this study,the main results are as follows:1.Mitochondrial transcription termination factor 1 was cloned on the A06 linkage group(Bna A06.m TERF1),which encodes 285 amino acids and contains 5 m TERF functional domains.37 m TERF family proteins were screened by bioinformatics analysis,and phylogenetic tree,gene structure and chromosome localization were analyzed in Brassica napus.2.The functional complementary expression vector of Bna A06.m TERF1 gene was constructed.the Arabidopsis m TERF1(soldat10)mutant was transformed,and the Arabidopsis phenotype returned to the wild type(Ler),indicating that the gene has a conserved function in rape and Arabidopsis.3.Analysis of the expression of Bna A06.m TERF1 in rape roots,stems,leaves(stalked leaves,sessile leaves),petals,different ovary lengths before flowering and ovary at different developmental stages after flowering by q RT-PCR,Bna A06.m TERF1 is expressed in various tissues.Among them,the expression level was highest in roots,followed by petals,and lowest in sessile leaves.4.Using CRISPR/Cas9 technology,two targets were designed respectively for two copies of rape m TERF1(one is specific to A06,and the other target can simultaneously knock out A06 and C07),and the knockout vector of m TERF1 was constructed and transformed into Zhongshuang 11 in Brassica napus.The results showed that:(1)16 edited lines of T0 generation were obtained,and 9 lines were characterized with the target gene on A06 chromosome be knocked out,while another 2 lines were edited for the gene on C07 chromosome.However,these 11 edited lines appeared no difference in leaf color from controlled lines(Zhongshuang 11).(2)5 lines were characterized for two copies of m TERF1 be knocked out,and obvious loss of chlorophyll in leaves of these 5 lines was investigated,(3)The relative chlorophyll content(SPAD)of the leaves of these 5 lines was determined,which also indicated that the chlorophyll content of the edited plants was lower than that of the control plants.In summary,the results suggested that the m TERF1 gene affect the development of plant chloroplasts.