Highly efficient CRISPR/Cas9-mediated targeted mutagenesis of multiple genes in Populus.

The typeⅡCRISPR/Cas9 system (Clustered regularly interspaced short palindromic repeats /CRISPR-associated 9) has been widely used in bacteria, yeast, animals and plants as a targeted genome editing technique. In previous work, we have successfully knocked out the endogenous phytoene dehydrogenase (PDS) gene in Populus tomentosa Carr. using this system.

Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge.

A cDNA encoding chalcone synthase (CHS), the key enzyme in flavonoid biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula Bunge by rapid amplification of cDNA ends (RACE). The full-length cDNA of S. viscidula CHS, designated as Svchs (GenBank accession no.

[Cloning and characterization of the PTS2 receptor gene (GhPex7) from cotton (Gossypium hirsutum L.)].

Peroxisomal targeting signals (PTS), including PTS1 and PTS2, were proposed to play an important role in introducing proteins into the peroxisomal matrix. Pex7, the PTS2 receptor, is crucial to the import of PTS2 proteins. Based on the sequence of a fragment (F010) recovered from cDNA-AFLP, the full-length cDNA sequence was obtained by RACE and the contig in cotton ESTs, and the coding sequence was further cloned.

[Cloning and characterization of a LRR resistance like (GhLRR-RL) protein gene from cotton (Gossypium hirsutum L.)].

LRR (Leucine rich repeat) proteins are involved in various biological processes. RACE was used to extend the 5' and 3' unknown sequence of a cDNA-AFLP fragment from cotton ovule, which had sequence similarity to the Arabidopsis thaliana LRR resistance-like (LRR-RL) protein. By joining the sequence of cDNA-AFLP fragment and its 3' and 5' RACE product, it was found that the full-length cDNA (GenBank Accession No.

[Cloning and characterization of D-113 gene promoter from cotton].

To study the expression of late embryogenesis abundant gene in seeds, the 1,024 bp 5' flanking sequence of D-113 gene, a late embryogenesis abundant gene of Gossypium hirsutum cv. Coker 312, was cloned by PCR. The similarity compared with the sequence of Lea protein gene family published was 92.

[PCR walking in cotton genome using YADE method].

A Y-shaped adaptor dependent extension (YADE) method was developed to amplify the unknown genomic sequences adjacent to a known sequence, with the single-primer amplification completely suppressed. Using this method, two fragments, F027S and F027A, adjacent to a cotton ovule cDNA fragment (F027), were amplified using genomic DNA. It was demonstrated that F027S and F027A had 104 bp and 175 bp overlapped to F027, and contained 1 and 3 putative introns, respectively, all of which included conserved border sequence GT-AG and a putative branch site.