CAJ | 학술논문

番木瓜果实采后贮藏期间的迅速软化与β-Gal基因的表达密切相关.利用常规PCR结合反向PCR技术,分离了一条4501 bp的番木瓜β-Gal基因组序列.其共含有17个内含子,外显子部分与相应的cDNA序列只有1个碱基的差异.生物信息学分析结果表明,该番木瓜β-GAL属于糖苷水解酶超级家族42中家族35的成员,在进化过程中与拟南芥的亲缘关系较近,与鳄梨和北美云杉则较远.同时,它还具有一段定位于胞外的信号肽,再次表明其可能参与了果肉细胞壁的降解和果实软化.进一步分离了β-Gal基因启动子,初步验证了其果实表达特异性.将该启动子替换载体p2301/TTRG上的CaMV 35S启动子,构建出RNAi-β-Gal双T-DNA植物表达载体.酶切分析和PCR检测结果表明,载体p2301/BPTTRG已被成功导入农杆菌,可用于后续的遗传转化研究.
번목과과실채후저장기간적신속연화여β-Gal기인적표체밀절상관.이용상규PCR결합반향PCR기술,분리료일조4501 bp적번목과β-Gal기인조서렬.기공함유17개내함자,외현자부분여상응적cDNA서렬지유1개감기적차이.생물신식학분석결과표명,해번목과β-GAL속우당감수해매초급가족42중가족35적성원,재진화과정중여의남개적친연관계교근,여악리화북미운삼칙교원.동시,타환구유일단정위우포외적신호태,재차표명기가능삼여료과육세포벽적강해화과실연화.진일보분리료β-Gal기인계동자,초보험증료기과실표체특이성.장해계동자체환재체p2301/TTRG상적CaMV 35S계동자,구건출RNAi-β-Gal쌍T-DNA식물표체재체.매절분석화PCR검측결과표명,재체p2301/BPTTRG이피성공도입농간균,가용우후속적유전전화연구.
Rapid softening of papaya fruit (Carica papaya L.) during postharvest storage is closely related to the expression of β-Gal gene. By using conventional and inverse PCR, a 4501 bp genonuc sequence coding for papaya β-Gal gene was isolated, containing 17 introns and exons with one base different from the cDNA sequence. Bioinformatic analysis of this gene revealed that this protein belonged to the member of fanuly 35 of glycoside hydrolyase superfamily 42, and had a closer genetic relationship with Arabidopsis thaliona, but farther with Persea american.a and Picea sitchensis. Additionally, the predicted protein included a signal peptide located extracellularly, indicating again the possible involvement of this enzyme in the degradation of cell wall matrix and fruit softening. The corresponding promoter of β-Gal was also isolated,and proved to be of expression specificity in the fruit. CaMV 35S promoter on the vector p2301/TTRG was replaced by β-Gal promoter, leading to the construction of β-Gal-RNAi Two-T-DNA plant expression vector p2301/BPITRG. The transformation of p2301/BPTTRG into Agrobactrium tumefaciens was confirmed by restriction enzyme analysis and PCR assay.