番茄红素β-环化酶基因(LcyB)启动子调控LcyB RNAi双元载体构建

Construction of RNAi Binary Vector to Silence Lycopene β-cyclase Gene LcyB Driven by Native Lcy B Promoter

根据番茄基因组DNA序列信息设计引物进行PCR扩增了Micro-Tom中番茄红素β-环化酶(Lycopeneβ-cyclase,Lcy B)基因起始密码子上游1 534 bp启动子区域序列(Lcy Bp),生物信息学分析表明,该启动子序列中存在TATA-盒、CAAT-盒、昼夜节律响应元件Circadian、光响应元件Box I、真菌激发子响应元件Box-W1、低温响应元件LTR、响应赤霉素的作用元件P-box、乙烯响应元件ERE、响应生长素的作用元件TGA-element等顺式作用元件.依据番茄Lcy B基因序列,设计2对含有不同酶切位点的特异引物进行PCR扩增Lcy B基因3’端特异的276 bp DNA片段,利用RNAi载体p KANNIBAL构建了"Lcy B启动子-Lcy B基因正义片段(Sense)-PDK内含子-Lcy B基因反义片段(Antisense)-OCS终止子"的RNAi表达框,并将这一RNAi表达框插入植物双元表达载体p ART27的Not I位点,构建成本研究的Lcy B启动子驱动的Lcy B基因RNAi植物双元表达载体p ART-Lcy Bp-RNAi-Lcy B.为利用RNAi技术特异性敲除Lcy B基因进而提高番茄果实中番茄红素含量奠定实验基础.

Suppressing the expression of gene encoding lycopene β-cyclase （ LcyB）, the key enzyme regulating the metabolism of lycopene to β-carotene, is an effective way to increase the content of lycopene. In the present study, a 1 534 bp promoter of 5＇ upstream of LcyB gene was cloned from Micro-Tom tomato （Lycopersicon esculentum） and bioinfomlatically analyzed in the database of plant cis-acting regulatory element （PlantCARE）. The result showed that several important cis-acting elements were identified in the promoter, including the TATA-box, CAAT-box, Circadian （response to circadian rhythm）, Box I （response to light）, Box-W1 （response to fungal elicitor）, LTR （response to low-temperature）, P-box （response to gibberllin）, ERE （response to ethylene） and TGA-element （response to auxin）. The LcyB promoter was inserted into the plasmid pKannibal between Mcr I and Xho I restriction sites to replace the CaMV 35S promoter, resulting in pK-LcyBp vector. Based on tomato genomic DNA sequence reported in the Gen- Bank （ Accession No. AEKE02020044）, two pairs of primers containing various restriction sites were designed to amplify the 3＇ terminal 276 bp fragment of LcyB gene from Micro-Tom tomato. The two 276 bp LcyB fragments were separately cloned into the plasmid pK-LcyBp between Xho I/Kpn I and Hind Ⅲ/Cla I sites, in sense or antisense directions, generating pK-LcyBp-RNAi-LcyB construct harboring LcyB RNA interfering expression frame driven by LcyB promoter. The RNA interfering expression frame was then cloned into the Not I site of the binary expression vector pART27 to achieve the RNAi binary vector targeting to silencing LcyB gene driven by its native promoter. This investigation lays the foundation for future increasing lycopene content through specific knock-out of LcyB gene by RNAi in tomato fruit.