Functionnal Study On Flowering Time Related Genes CO,C3H17 And KHZ1 In Brassica Napus

Canola is an important oil crop in China.Flowering time is a key character in rapeseed breeding because it directly affects the maturity of rapeseed.The flowering time of rapeseed is affected by the two major factors:temperature and photoperiod.Photoperiod is the key factor of flowering regulation,and the same season varies little from year to year.With the development of molecular technology and the discovery of more and more gene functions in Arabidopsis thaliana,all of the genetic information could be used to analyze the function of homologous genes in Brassica napus.In this study,Bna CO,Bna C3H17 and Bna KHZ1 were selected as the target genes according to the homologous comparison with A.thaliana.CO（CONSTANS）is an important transcriptional regulator in the photoperiodic pathway.C3H17 and KHZ1 are CCCH family genes in Arabidopsis,which play important roles in plant growth and stress.Overexpression vectors of these genes were constructed to transform A.thaliana and multi-target CRISPR/Cas9 vectors to transform B.napus variety Westar.The phenotypes of overexpressed lines and mutants were investigated.The main results are as follows:1. It was found that both copies of Bna CO,Bna C09.CO and Bna A10.CO,could cause early flowering in the overexpressing A.thaliana lines,respectively.Compared with the wild type,the flowering days of the overexpressed lines were 3-5 days earlier and the rosette leaves were 3-4 fewer.Through q RT-PCR,the relative expressions of Bna C09.CO,Bna A10.CO and its downstream genes FT and SOC1 were significantly higher than those of the wild type.It indicated that Bna C09.CO and Bna A10.CO played a similar role to CO,which could upregulate the expression of FT and SOC1 in A.thaliana to promote flowering differentiation under long-day condition.2. The multi-target CRISPR/Cas9 vectors were constructed by designing 1-4 sg RNA for each copy of three genes.The positive rates of transgenic plants of Bna CO,Bna C3H17and Bna KHZ1 were 57.69%,56.25%and 47.19%,respectively.In the T₀ generations,the fourth target of each vectors had high efficiencies,which distributed between 43.75%-100.00%.Among the various mutation types,single base insertion was the main mutation type,and the mutation mainly occurred at 3bp upstream of PAM.The homozygous mutation rate was low for the whole T₀ generations and the biallele mutation rate of transgenic plants of Bna C3H17 was high,reaching 64.71%.3. In the investigation of T₁ generation mutants,a homozygous double mutant onBna C3H17 was identified,and found that the flowering time of the line was delayed by 7days compared with Westar.Similarly,the flowering time of a double mutant on Bna CO line was more than 7 days later than that of Westar.A mutant line edited in four copies of Bna KHZ1 was identified,and the flowering time of this line was also slightly later than that of Westar.In summary,Bna CO could promote the flowering differentiation in B.napus.It was also showed that Bna C3H17 and Bna KHZ1 had some effect on the flowering differentiation in B.napus.Further research must to be done.Other features of Bna C3H17 and Bna KHZ1remained to be explored.