Screening And Functional Identification Of Genes Responding To Abiotic Stress Based On The FOX Hunting System In Brassica Napus L.

Brassica napus,the main crop of Northern Europe,Canada and China,is the one of most important oil crops in the world,accounting for 16% of the world's total vegetable oil production.With the steady growth of global vegetable oil consumption,the annual growth rate is about 5%.Brassica crops are susceptible to osmotic stress,such as drought and salinity.Facing the steady growth of vegetable oil consumption and the poor growth conditions,it is urgent to develop the resistant Brassica napus to maintain yield stability under adverse environmental conditions.Over the past few decades,transcription factors,responding to various stress,have been used to explore the stress tolerance of plants by transgenic methods.Previously,we collected the transcripts from Brassica napus treated by drought.Base on the high-throughput sequencing,the full-length cDNAs of differentially expressed genes were screened and cloned.An plant expression library containing 70 drought response genes was constructed by FOX hunting system(full-length cDNA overexpressor gene hunting)and high-throughput Gateway technology.And then the expression library was transformed into Arabidopsis to obtain the library of the Brassica napus FOX Arabidopsis.We screened lines obtained phenotypes from the transformants with different treatment stresses,followed by sequencing the genes inserted in 125 transformants and performing the next functional verification.1.The homozygous lines were screened from 125 lines.And 42 homozygous lines were obtained in T3 or T4.2.The stress resistance analysis and growth phenotypes monitoring were performed for the obtained 42 homozygous lines.Combined with the function of the gene we observed,we selected four genes: CONSTANS LIKE gene family BnCONSTANS-LIKE2(BnCOL2)and genes of NAC family BnNAC18,BnNAC1-1,BnNAC5-11.And then we performed the next step to identify the function.3.BnCOL2 has transactivational activity and is localized in the nucleus.The tissue expression analysis showed that BnCOL2 expressed preferentially in the cotyledon,especially in leaves.Expression patterns indicated that BnCOL2 was induced by NaCl,heat,cold,especially PEG and ABA.BnCOL2 overexpressing Arabidopsis showed sensitive phenotypes compared with wild type Arabidopsis under various stress conditions.In BnCOL2 overexpression Arabidopsis,the rates of the mortality after drought treatment,leaf water loss and stomatal density were higher than control.And The leaf temperature was lower.The chlorophyll content was higher and the bolting time was earlier.4.BnNAC1-1,BnNAC5-11 and BnNAC18 have transactivational activity.By subcellular localization,it was found that the three BnNACs were located in the nucleus.The BnNAC1-1 was expressed in root and cotyledon,BnNAC5-11 was expressed preferentially in the cotyledon,and BnNAC18 was expressed in cotyledon and leave by tissue expression analysis.Expression patterns indicated that the three genes were induced by NaCl,heat,cold,especially PEG and ABA.5.CRISPR/Cas9 vectors were constructed for the four genes.And then CRISPR/Cas9 vectors and overexpression vectors were introduced into the Brassica napus,and transformants had been obtained.