| Terpenoids are a sort of main secondary metabolites in plants, and play important roles in plant growth, development and defense. Currently, many studies about plant terpenoids metabolism regulation are based on a comprehensive understanding of the key enzymes in the metabolic pathway. As an important ratelimiting enzyme in the upstream of MVA pathway, it is necessary to analyse HMGR gene family. Apple is a kind of important cash crop in China, the analysis of HMGR genes in the apple genome has not been reported yet. Apple genome was successfully sequenced in 2010, on this basis, this study launch a general bioinformatics analysis of the HMGR genes family in apple genome. Moreover, we studied the expression profiles about these genes by RT-PCR technology. Furthermore, In order to study the HMGR1 L gene function, we constructed a HMGR1 L expression vector and transformed into Nicotiana benthamiana.The main results are as follows:(1) By using Blastp and Structure domain filtering, we obtain eight HMGR genes in apple genome. They named as HMGR1, HMGR2, HMGR3, HMGR1 L, HMGR5, HMGR6, HMGR7, HMGR8, respectively. Compared with other species, apple HMGR genes familiy is larger. Analysis show that the secondary structure of apple HMGR protein mainly comprized by Alpha helix and random coil, tertiary structure appears as a "V" shape.(2) Chromosomal location analysis showed that apple HMGR genes unevenly distribute on the 5 apple chromosomes. HMGR3 and HMGR6 located on chromosome 2, HMGR8 located on chromosome 8, HMGR2 located on chromosome 9, HMGR1 L and HMGR5 located on chromosome 10, and HMGR7 located on chromosome 16.(3) Evolution analysis showed that apple HMGR proteins were divided into 3 groups. HMGR1, HMGR1 L, HMGR5, HMGR7 clustering for the first group. HMGR2, HMGR6 clustering for the second group. HMGR3, HMGR8 clustering for the third group. Gene structure analysis showed that apple HMGR gene consists of 2-6 exons. The gene structure of HMGR1, HMGR1 L and HMGR5 were similar. Besides, there are four conservative motif in apple HMGR protein, EMPVGYVQIP and TTEGCLVA are two HMG-Co A binding motifs, DAMGMNM and GTVGGGT are two NADP(H) binding motifs. During evolution, the GTVGGGT NADP(H) binding motifs has lower conservative than the other three.(4) There are two pairs of duplicated genes in apple genome. Among them, HMGR1/HMGR1 L were generated by fragment duplication, HMGR1L/HMGR1 L genes were generated by tandemt duplication, which suggest that fragment replication and tandem repeat events contribute to HMGR gene amplification in apple genome.(5) Tissue expression pattern analysis indicated that, besides HMGR6, the other seven apple HMGR genes were highly expressed in fruit, which implies they are closely related to fruit development. In addition, the organization expression patterns of HMGR1/HMGR1 L duplicated genes were similar, suggesting that they may participate in the same apple growth regulation process.(6) There are many cis-acting elements response to drought, low temperature, high temperature and wounding in apple HMGR gene promoter. Stress response profile analysis showed that the exprssion of apple HMGR genes were induced by damage and low temperature.(7) pBI121+Ht+HMGR1L expression vector were constructed and transformed into Nicotiana benthamiana. The transgenic plants were identified on genomic and transcriptional level, respectively. The result proved that we obtained transgenic lines successfully.(8) The expression of HMGR1 L in the root was significantly higher than other parts in transgenic tobacco. Overexpression of HMGR1 L promote the tobacco root development and the product synthesis in the MVA and MEP pathway, such as chlorophylls, carotenoids, brassinolide, abscisic acid and volatile components.(9) After 4? treatment, the transgenetic plants showed lower reactive oxygen contents, less membrane damage, higher SOD, APX activity and photosynthetic rate. These results showed that overexpression HMGR1 L increased the defense ability of transgenic tobacco under low temperature. |
PDF Full Text Request