Cloning And Functional Analysis Of U6 Promoters In Cotton And Creation Of Arabidopsis GGB Mutant

CRISPR/Cas9 genome editing technology has been successfully applied in many kinds of crops, but it was not reported that the CRISPR/Cas9 gene editing technology has been applied in cotton molecular breeding. U6 promoter is an important element for the transcription of sg RNA in the CRISPR/Cas9 genome editing system. There were at least ten kinds of U6 RNAs corresponding to U6 promoters in the cotton genome, whether they had transcriptional function and whether they had high transcription activity in cotton reproductive cells(pollen) were not clear;Previous studies showed that GGB gene in Arabidopsis had drought resistance. Whether the cotton GGB gene has the same drought resistance as the Arabidopsis GGB gene, and can be used as an ideal target for drought resistant breeding of cotton. The most reliable evidence comes from whether the cotton GGB functions can complement to the phenotype of Arabidopsis ggb mutants. In this study, several candidate U6 promoters were cloned to provide an ideal promoter for the construction of cotton CRISPR/Cas9 genome editing vector;At the same time, the CRISPR/Cas9 genome editing technique was used to obtain the Arabidopsis ggb mutant, which provided an ideal complement for the verification of the function of GGB gene in cotton. The main results were as follows:1. U6 promoter is an important element for the transcription of sg RNA in the CRISPR/Cas9 genome editing system. Five Gb U6 promoters were cloned from cotton variety Xinhai16(Gossypium barbadense L.), their length were 1 166 bp, 1 119 bp, 1 134 bp, 1 214 bp and 1 176 bp, respectively, and constructed five kinds of fusion expression vectors of Gb U6::GUS. After sequence comparation of U6 promoter between Arabidopsis and cotton, results showed that cotton U6 promoter contained the conserved-60 USE motif and-30 TATA box and the distance between two elements also was fixed, just like Arabidopsis U6 promoter. The results of transient transformation with particle gun showed that four cloned Gb U6 promoters could drive the expression of GUS gene in cotton pollen which were stained into blue. Among the four promoters, the Gb U6-5P::GUS exhibited deeper blue, similar to the Ca MV35 S promoter.2. According to the sequence of Gb U6-5P promoter cloned, six different truncated U6 promoters were successfully cloned using Transfer PCR method and their length were 672, 468, 358, 280, 202 and 105 bp, respectively. GUS fusion expression vectors driven by corresponding truncated promoter were constructed and transformed into cotton pollen by the vacuum in filtration transformation method. Results of GUS histochemical staining showed that the cloned seven truncated Gb U6-5P promoters could drive GUS expression in cotton pollen and all corresponding cotton pollen could be stained blue, but there exist different shades among them. Results showed that the shorter promoter, the stronger transcription activity, and so do the two other Gb U6 promoter. GUS staining results showed that the shorter U6 promoter had higher transcriptional activity, and the characteristics on different U6 promoter had in common.3. GGB is a negative regulator of drought resistance. The editing vector with GGB gene target sequence was constructed and transferred into Arabidopsis by Agrobacterium-mediated floral dip. After sequencing of GGB in T2 generation, two kinds of mutants with a deletion of 4 bases and an addition of 1 base(T) were found at the target site respectively. Semi-quantitative RT-PCR analysis results showed that the expression of GGB gene was almost not detected in the ggb mutant, which indicated that the mutants were GGB knockout lines. Through measuring the water loss rate, drought resistant and seed yield per plant, ggb mutant exhibited decreased water loss rate, improved drought resistance but unchanged seed yield per plant compared to wild-type.