Genome-wide identification of LEA gene family and cold response mechanism of BcLEA4-7 and BcLEA4-18 in non-heading Chinese cabbage [Brassica campestris (syn. Brassica rapa) ssp. chinensis]

Cold stress is a key factor limiting the yield and quality of non-heading Chinese cabbage. The hydrophilic protective protein LEA plays an important role in plant abiotic stress. In this study, 72 BcLEAs were identified from non-heading Chinese cabbage and divided into 9 subfamilies by phylogenetic analysis. Gene structure analysis showed that BcLEAs were unevenly distributed on 10 chromosomes, with few introns. Through analyzing the expression of these genes under cold stress by RNA-seq and qRT-PCR, two genes (BcLEA4-7 and BcLEA4-18) highly sensitive to cold stress were identified, whose roles in cold tolerance of non-heading Chinese cabbage were demonstrated by virus-induced gene silencing. The BcLEA promoters were analyzed to study the cold response mechanism of BcLEA4-7 and BcLEA4-18, revealing that both BcLEA4-7 and BcLEA4-18 promoters contained two CRT/DRE elements. Subsequently, it was found that the promoters isolated from non-heading Chinese cabbage could be activated at low temperatures. Further analysis showed BcCBF2 in non-heading Chinese cabbage interacted with two CRT/DRE elements in BcLEA4-7 and BcLEA4-18 promoters to stimulate their activity, indicating that BcCBF2 is an upstream regulator. Meanwhile, the CRT/DRE element located in BcLEA4-7 promoter (-219 bp to-171 bp) and BcLEA4-18 promoter (-234 bp to-186 bp) was more likely to be activated by BcCBF2, which may be attributed to its flanking sequence. These data laid a foundation for further under -standing the functional role and regulatory mechanism of BcLEAs in cold stress tolerance.

Automated summary

This study identified 72 BcLEAs from non-heading Chinese cabbage and divided them into 9 subfamilies. Gene structure analysis showed that BcLEAs were unevenly distributed on 10 chromosomes. Two genes (BcLEA4-7 and BcLEA4-18) highly sensitive to cold stress were identified and their roles in cold tolerance were demonstrated. The promoters of BcLEA4-7 and BcLEA4-18 contained two CRT/DRE elements and could be activated at low temperatures. BcCBF2 interacted with the CRT/DRE elements in these promoters to stimulate their activity, indicating BcCBF2 as an upstream regulator.