Creation Of Thermoreceptor Genes Mutants For Pol TCMS In Brassica Napus L.

Polima cytoplasmic male sterile line（pol CMS）is the most commonly used male sterile line in rapeseed hybrid production in China,among which Polima cytoplasmic temperature-sensitive male sterile line（pol TCMS）as a model of two-line breeding has the characteristics of fertility and temperature control.The sterile line partially regained its fertility at a low temperature of 16°C and was completely sterile at a high temperature of24°C,but the key genes and molecular mechanisms of thermosensitive sterility in the pol TCMS are unknown.In this study,four reported Arabidopsis thermoreceptor genes（At PHYB,At PIF4,At ELF3 and At ARP6）were used to study temperature-sensitive sterility,and CRISPR/Cas9 gene editing technology was used to create mutants of related thermoreceptor genes.The main results of this study are as follows:1.Temperature-sensitive phenotyping of pol TCMS of cabbage-type rapeseed.The high temperature induction time of high temperature 24°C with different time gradients after entering the flowering stage was carried out before the flowering stage,and the high temperature induction time leading to complete sterility of pol TCMS must reach 40 min.When the low-temperature 16℃pre-cultured materials were transferred to high-temperature 24°C culture,the buds of different sizes were morphologically marked and the fertility of each bud after flowering was observed,and the observation results showed that the buds with pol TCMS less than 1 mm showed complete male sterility after induction by high temperature.2.Bioinformatics analysis of temperature receptor genes.We identified 11 Bna PHYs,23 Bna PIFs,14 Bna ELFs and 28 Bna ARPs from the ZS11 genome of Brassica napus L.The homologous genes of Bna PHYB,Bna PIF4,Bna ELF3 and Bna ARP6 showed 4,5,4and 3 homologous copies,respectively.Evolutionary analysis showed that Bna PHYB,Bna PIF4,Bna ELF3 and Bna ARP6 were highly homologous to At PHYB,At PIF4,At ELF3and At ARP6,respectively.3.In Brassica napus L.ZS11,the expression level of Bna A05.PHYB was higher in the bud and upper stem,and the expression level of Bna A05.PHYB in 2 mm bud was 2.42 times that in 4 mm bud.The expression of Bna C03.PIF4 was higher in 2 mm bud and upper stem.Bna ELF3 was mainly expressed in the upper stem,and Bna C08.ELF3 was the highest.The expression of Bna ARP6.C03 was higher in 2mm bud and upper stem.Gene expression analysis showed that Bna AO5.PHYB,Bna C08.ELF3 and Bna C03.ARP6 were up-regulated and Bna PIF4 was down-regulated under 24℃.4.A total of 94 single gene mutants were obtained from T₀generation,including 31phyb mutants,20 pif4 mutants,15 elf3 mutants and 14 arp6 mutants.The editing efficiencies of Bna PHYB,Bna PIF4,Bna ELF3 and Bna ARP6 were 92.31%,66.67%,55.56%and 58.82%,respectively,for CRISPR/Cas9 positive plants of T₀ generation.Different techniques of editing have their own efficiencies like deletion（16.09%）,Complex mutation（32.87%）,Insertion（50.22%）and replacement（0.82%）.Meanwhile,the mutation probability of A/T base pair was higher than that of C/G base pair in single base mutation.5.Investigation of mutant phenotype.Bna PHYB may affect the fertility recovery of pol TCMS at low temperature.At low temperature of 16℃,the fertility of phyb mutant was different from that of wild type 596.The phyb mutant showed inhibited filament elongation,significantly less pollen and reduced petal size.In addition,the results showed that the phyb mutant showed reduced leaf size and branch number during growth.In summary,it is speculated that Bna PHYB affects the development of cabbage-type rapeseed plant type and may participate in regulating the fertility recovery of pol TCMS at low temperature.No fertility changes were observed in mutants of other genes in this study,and their function needs to be further studied.