Aldehyde Reductases Gene In Brassica Oleracea: Response To The Infestation By Plutella Xylostella And Construction Of Its Transgenic Plant

Aldo-keto reductases (AKRs) metabolize exogenous toxic substances and play a key role in protecting plants from abiotic stress. It was found that Brassica oleracea released aldehydes under the no-damage or mechanical-damage situation, but released alcohols when attacked by Plutella xylostella in our laboratory. Therefore, aldo-keto reductases may play an important role in plant defense against insects.The relative expression levels of AKRs in the B. oleracea were detected by qPCR. The results showed that the relative expression levels of BoAKR1 and BoAKR2 were significantly increased 2 h after the feeding of P. xylostella and reached the maximum 10 h after feeding, which indicated that BoAKR1 and BoAKR2 in B. oleracea positively responded to the infestation by P. xylostella. After mechanical injury, the expression of BoAKR1 and BoAKR2 was significantly increased immediately, and BoAKR1 reached the maximum in 4 h, and BoAKR2 reached the maximum immediately after the damage. It indicated that BoAKR1 and BoAKR2 could respond to mechanical injury with a stronger response by BoAKR1 than by BoAKR1. The expression levels of BoAKR3 and BoAKR4 were not significantly changed after the mechanical damage and the expressions of BoAKR3 and BoAKR4 were significantly increased 10 h after P. xylostella feeding, which indicated that BoAKR3 and BoAKR4 did not respond to the mechanical injury, but the infestation by P. xylostella. The expression of BoAKR5 was not significantly changed either by the mechanical injury or by the infestation of P. xylostella, which meant that BoAKR5 might not be involved in the defense against the mechanical injury and the infestation by P. xylostella.BoAKR1 was linked to the CaMV35S promoter in pCAMBIA1301 plasmid to construct the over-expression vector. BoAKR1(+) and BoAKR1(-) were firstly inserted into the pJM007 plasmid to construct the clone vector of pJM007+RNAi(BoAKR1), and then the RNAi construct (promoter+BoAKR1(-)+intron+BoAKR1(+)+terminator) from the clone vector was inserted into pCAMBIA1301 plasmid to construct theBoAKR1 RNAi plant vector. A gRNA target for BoAKR1 was designed and then inserted into the VK005-08 plasmid to produce the CRISPR/Cas9 for BoAKR1.The seed contamination rate in order was 4%NaClO>6% NaCIO>8% NaClO> 10% NaCIO=12% NaCIO. Therefore,10% NaClO was recommended. Six kinds of callus induction medium were tested and the results showed that MS+3.0 mg/L 6-BA +0.45 mg/L NAA+3% sugar+0.7% agar powder was most efficient for the regeneration of Brassica oleracea. Callus were obviously generated on the 7th day and 24 cotyledons were induced into callus after 20 d.This study provides a foundation for the defensive role of AKR gene in B. oleracea against P. xylostella.