Cloning and Functional Analysis of Different Truncated U3 Promoters in Tomato

doi:10.7668/hbnxb.201750912

Abstract

Abstract: It is necessary for us to clone some endogenous promoters with high transcription activity in tomato leaves and construct CRISPR/Cas9 system, which could provide an effective technology for the research of molecular design breeding in tomato. Six different truncated tomato U3 promoters had cloned by two rounds of PCR amplification from tomato variety Zhongshu 4. Six GUS fusion expression vectors were constructed and transformed into tomato leaves by the agroinfiltration. After right sequencing of the transfer PCR products, the necessary element for transcriptional function of six kinds of SlU3 promoter was analyzed using DNAMAN software. The different truncated SlU3-1P,SlU3-3P and SlU3-4P promoters from tomato variety Zhongshu 4 were performed to clone by two rounds of PCR amplification, altogether there were six different promoters. Lengths were 489,318,450,248,457,248 bp respectively. Six GUS fusion expression vectors were constructed and transformed into tomato leaves.The promoter sequence alignment analysis revealed that the tomato U3 promoter, like the Arabidopsis U3 promoter, also contained two more conserved elements, the USE and TATA boxes, and the positions between the two elements were relatively fixed.Transfected tomato leaves by agroinfiltration transformation method. The results showed that the six truncated U3 promoters could drive GUS expression in tomato leaves and tomato leaves could be stained blue and the shorter promoters also had stronger transcription activity as original promoters.Six different tomato U3 promoters had cloned with high-level transcription in tomato leaves, which could provide ideal endogenetic promoters for the construction of CRISPR/Cas9 system for tomato functional genomics research.

Key words: Tomato, SlU3 promoters, Clone, Agroinfiltration, Tomato leaves

CLC Number:

PU Yan, LIU Xiaodong, AERZU GULI·Ta shi, WEI Qian, LIU Chao. Cloning and Functional Analysis of Different Truncated U3 Promoters in Tomato[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(1): 33-39. doi: 10.7668/hbnxb.201750912.

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