The Analysis Of Functional Difference Of BaPDS1 And BaPDS2 In Chinese Kale

Chinese kale?Brassica oleracea var.alboglabra?is a vegetable of the Brassicaceae,with the tender buds or young leaves as the main edible organs.The vegetables has rich nutrients including vitamin C,carotenoids,glucosinolates and polyphenols,therefore they are loved and greeted by consumers.Phytoene desaturase is one of the key rate-limiting enzymes in the carotenoid biosynthesis pathway.We found that the PDS gene has two members in Chinese kale in our early experiments,but the function and relationship between them are not yet clear.Therefore,the systematic analysis of the functional differences of BaPDSs gene family members,through spatio-temporal expression,the light and hormone were selected to perform treatment on Chinese kale seedlings according to the results of promoter cis-element analysis,subcellular localization,CRISPR knockout mutant phenotype,using white flower Chinese kale as experimental material.This study will provide a theoretical basis to further improve the content of carotenoids and the quality of nutrition in Chinese kale.The main contents and results are as follows:1.The two members of the PDS gene family encoding phytoene dehydrogenase?PDS?in the Chinese kale were successfully isolated by reverse transcription PCR and the specific primers were designed by homologous sequence analysis,and named as BaPDS1and BaPDS2,respectively.They were deposited in NCBI with accession number KX426039 and KX426040 respectively.The length of the coding sequence and the number of encoded amino acids of these two genes were 1 692 bp,563 and 1 698 bp,and 565,respectively.According to the analysis of homology and the construction of evolutionary tree,the BaPDSs protein is closely related to Brassica oleracea,Brassica juncea,Brassica rapa,Arabidopsis thaliana and other plants of the Brassicaceae.At the same time,it was found that PDS protein is more conservative during evolution.2.The spatio-temporal expression analysis of Chinese kale showed that the expression of PDS genes differ significantly at both temporal and spatial levels.The relative expression levels of BaPDSs were lowerin germinating seeds,and then sharply induced thereafter.BaPDS1 was constitutively expressed in different organs of mature stage,while BaPDS2 abundance was obviously different among organs at the same developmental stage.Particularly,no BaPDS2 was detected in young seeds.The transcription levelsof BaPDSs were significantly lower in sepals than in other flower tissues.BaPDS1 was accumulated in stamens and pistils during blooming.3.The promoters of BaPDS1 and BaPDS2 were isolated to perform cis-acting element analysis,and were counted the similar and different cis-elements,respectively.Light and hormone were selected to perform three different treatments on Chinese kale seedlings according to the prediction results.The results showed that the BaPDS1 gene responded to blue light and red-blue lights,blue light promoted BaPDS1 expression,peaked at 3 h,and decreased in the later phase,BaPDS1 expression was significantly higher than the control under red-blue light irradiation,BaPDS2 only responds to blue light,but blue light is observed to inhibit its expressionand under the different light quality treatments.The strong light induced increased expression of BaPDS1 and peaked at 24 h,whereas the weak light induced increased the expression of BaPDS2,peaked at 12 h,and dark suppressed BaPDS2 expression and the amount of expression gradually tends to 0under the different light intensity treatments.Treatments of the different hormone,BaPDS1 and BaPDS2 co-reacted with SA?ABA and MeJA,in addition,BaPDS2 also responded to GA3.The expression levels of BaPDS1 and BaPDS2 were showed the tendency declining at the begining and rising up in late,and the expression were significantly higher than that of the control in the ABA treatment.The expression levels of BaPDS1 and BaPDS2 were significantly lower than the control in the SA treatment.In GA₃ treatment,the expression of BaPDS1 has not significantly different compared to the control,but the expression of BaPDS2 peaked at 1 h,and the expression was lower than the control after 1 h.In MeJA treatment,the expression of BaPDS1 was basically lower than control,while the expression of BaPDS2 was significantly higher than the control,except for 12 h.4.The subcellular localization vectors pC2300-35S-BaPDS1-eGPF and pC2300-35S-BaPDS2-eGPF were constructed and transferred to the protoplasts of Chinese kale.It was observed that both BaPDS1 and BaPDS2 were located on the chloroplast using fluorescence microscope,which was consistent with the prediction.5.The BaPDS1 and BaPDS2 genes were selected as the common target gene,and the CRISPR/Cas9 vector?pCC-Target-sgRNA?was constructed and transferred into Agrobacterium tumefaciens-leafed cotyledons to obtain homozygous transgenic plants with double and/or single mutations.The observation of plant phenotypes and the determination of color difference showed that the plants exhibited the albino phenotype regardless of single or double mutants,but double mutants had more obviously pure albino phenomenon,indicating that the two genes BaPDS1 and BaPDS2 are equally important to the growth and development of Chinese kale,and the lack of any one will hinder the normal synthesis of carotenoids and chlorophyll,resulting in the obstruction of the growth and development of the plant,and ultimately leading to albino and death.The results showed that BaPDS1 and BaPDS2 genes play different roles in the carotenoid biosynthesis in Chinese kale,while their functions are still partially complementary.