关键词: 草莓, 果胶裂解酶, amiRNA干扰, P119果实特异性启动子

Abstract: Pectate lyase(PL) is one of the most profitable candidate enzymes for improving the fruit firmness and delaying the softening process in strawberry(Fragaria×ananassa Duch.) by molecular modifying. The role of PL in strawberry fruit softening was studied by amiRNA-mediated silencing. The changes of pectate lyase activity and fruit firmness were determined across the fruit development of cultivated strawberry ‘Benihoppe’. Candidate PLs were submitted toWMD3(http://wmd3.weigelword.org) for miRNA target site screening(target Fragaria×ananassa PUT v183, minimum number of included targets:one). Those candidate miRNA target sites were then manually checked, ensuring that the target loci exist in all PL transcripts. This target sequence was introduced into the Arabidopsis thaliana miR390a precursor sequence as the backbone. The validated construct was transformed into the Agrobacterium GV3101. Transient expression in strawberry fruits was conducted by agroinfilltration. Expression level of the PL genes was monitored by RT-PCR. Fruit firmness was examined too. With the development of strawberry fruit, the PL activity gradually increased, especially the stages after turning red. The PL activity increased sharply and reached the maximum in the full red period. In contrast, fruit firmness gradually decreased during fruit development especially during the process from big green to color transition, and then the rate of hardness reduction slowed. The P119 promoter from tomato was able to drive amiRNA gene expression, which can reduce the expression of PL gene in strawberry fruit by 34.5%. The knock down of PL gene does not interfere with the accumulation of anthocyanins in strawberry, and the silencing group had higher fruit hardness than the control group. In a comprehensive analysis, PL regulates the dissociation of cell wall mainly in the late fruit developmental stage. Obviously, from our results, there are also other factors that affect the softening of strawberry. The p119 fruit-specific promoter has transcriptional activity in silencing PL gene of strawberry in the fruit, which lays a foundation with the application in future molecular modification. To this end, stable transformation is underway. Therefore, PL regulates plants cell wall dissociation at the late development stages of strawberry. Silencing the gene with P119 using amiRNA can delay fruit softening process.

Key words: strawberry, Pectate lyase, AmiRNA, P119 fruit-specific promoter