Gene Expression Analysis And Mutant Creation Of Floweing-Time Gene BnaFRI In Brassica Napus

Flowering is an important process in plant development.Adaption of flowering to climate fluctuation can ensure high and stable yield of crops.In Arabidopsis thaliana,FRIGIDA（FRI）and FLOWERING LOCUS C（FLC）are the key genes to regulate the flowering time and to determine the eco-types.FRI can promote FLC expression while FLC can represses the expression of FLOWERING LOCUS T（FT）and results in the late flowering.Brassica napus could be classified as spring type,semi-winter type and winter type.There are four BnaFRI paralogues in genome of B.napus.Previously studies had revealed that the sequence variation of BnaFRI.A3 is related to the adaption and ecotype differentiation.In this study,the expression patterns of four BnaFRI paralogues in different ecotypes of B.napus were analyzed to explore the potential co-expression relationships with BnaFLC genes.Meanwhile,the mutants of BnaFRI paralogues were developed using CRISPR/Cas9 technology to dissect the role of BnaFRI genes in flowering regulation network of B.napus.The main results are described as follows:1.The expression patterns of four BnaFRI paralogues were analyzed in root,stem,leaf,stamen,pistil,flower,calyx and silique of Zhongshuang 11,which is a semi-winter type rapeseed of China.It was found that four BnaFRI genes were expressed in all eight tissues.BnaFRI.A3 and BnaFRI.C3 had highest expression level in stamens and flowers,and the expression level of BnaFRI.A3 was about two fold than that of BnaFRI.C3.The expression level of BnaFRI.A10 was much lower in stamens and flowers,however much higher in roots;BnaFRI.C9 was expressed in all tissues,and the expression level was all much lower.2.The dynamic expression patterns of BnaFRI genes and downstream BnaFLC genes were analyzed.Four BnaFRI paralogues were expressed in leaves from T0 to T4 throughout the flowering process of rapeseed.The expression level increased when vernalization began,and the peak appeared at T2 or T3.After vernalization,the expression levels of BnaFRI paralogues from decreasing to increasing.The expression levels of four downstream BnaFLC genes were decreased when low temperature appeared,however,the dynamic expression profile of different copies showed significant differences.There was no significant correlation of co-expression between four BnaFRI paralogues and four BnaFLC genes,BnaFLC.A2,BnaFLC.A10,BnaFLC.A3.a and BnaFLC.C2.3.The expression patterns of BnaFRI genes under different temperature were analyzed using qRT-PCR assay.In Zhongshuang 11,the similar expression profiles of four BnaFRI paralogues were found.With the temperature falling,the expression level increased first,then began to decrease when the temperature dropped to 10℃.The different expression pattens of four BnaFRI paralogues were found in Westar.With the decrease of temperature,the expression levels of four BnaFRI genes decreased first,then began to increased when the temperature dropped to 10℃.Compared with the expression profiles of BnaFRI paralogues,there was no significant correlation of co-expression between BnaFRI paralogues and two BnaFLC genes,BnaFLC.A2,BnaFLC.A3.a.4.Based on the tobacco transient expression system,the nuclear localization protein of BnaFLC.A10 in B.napus was selected as control,subcellular localization analysis showed that four BnaFRI proteins were located in the nucleus.5.In order to obtain mutants of four BnaFRI paralogues in Tapidor（winter type rapeseed）,CRISPR/Csa9 technology was selected.There were 30 plants of T₀ generation were obtained,and 8 plants were positive using PCR for detection.Sequence comparison between wild type and positive plants,s equence variations were only found in 2 plants,and mutation type were all 1 bp insertions in the first exon of BnaFRI.A3.Two mutations led to the fifth amino acid of BnaFRI.A3 produce a stop codon.No sequence variations was det ected in other positive plants.