Cloning And Function Analysis Of LPAT4 And LPAT5 Gene From Brassica Napus

Brassica napus as important economic crops in the world, is an important source of edible vegetable oil and high-quality protein. Data show that rapeseed oil content increase in 1%, the equivalent yield of rapeseed seeds 2.5% for oil production per unit area, thereby increasing rape seed oil content is an important way to improve production efficiency. LPAT is an important enzyme in the TAG Assembly process, which can affect lipid synthesis. Improving LPAT activity can reduce the inhibitory action in synthesis process. Thus, Cloning and function analysis function of LPAT gene by means of genetic engineering have certain practical significance of studing of oil content of rapeseed seeds.The major experiment conclusions are summarized as follow.1. Through the technology of homology-based cloning, two copies of LPAT4 full-length CDS sequences (1143 bp and 1140 bp) were cloned in this study, designated as BnLPAT4-1 and BnLPAT4-2, respectively. Bioinformatics analysis revealed that they shared the LPLAT_LCLAT1 like domain and belonged to the LPLAT superfamily.2. One copy of BnLPAT4 promoter was obtained by the technology of homology-based cloning. Element analysis show this promoter may involved in light responsiveness, hypoxia responsiveness, stress responsiveness, fungal responsiveness, abscisic acid responsiveness, gibberellin responsiveness, salicylic acid responsiveness, ethylene responsiveness, shoot specific expression and regulation of circadian rhythms. The expression result of GUSA gene driven by pBnLPAT4 indicate that the gene drived by this promoter mostly express in seeding, root, stem, petal, calyx,receptacles, capsules, while it is inactive in anther and seeds.3. Temporal and spatial expression results showed that BnLPAT4-1 and BnLPAT4-2 were ubiquitous expression genes. Among them, the highest expression of BnLPAT4-1 was in leaf, while the BnLPAT4-2 was in embryo. Stress analysis indicated that BnLPATA-1 and BnLPAT4-2 presented different expression patterns under the treatments of NaCl, PEG4000, waterlogging,6-BA and ABA. Pole difference analysis displayed that ABA had a great effect on the expression of BnLPAT4-1, while BnLPAT4-2’s expression was sensitive to PEG4000.4. Yeast expression of BnLPAT4-1 and BnLPAT4-2 presented that their expression product have transferase activity. The content of saturated fatty acid in BnLPAT4-1 transformant is 66.88%, it is 2.89 times of unsaturated one. In terms of the level of saturated fatty acid, it is 1.61 times higher than the 25.62% of YES2.0 transformant. BnLPAT4-2 transformants improved the ratio of C16 fatty acid(palmitic acid and palmitoleic acid) significantly. It is 22.40%higher than 25.62% for the level of C16 fatty acid in YES 2.0 transformant, approximately improved 70%.5. T3 Arabidopsis thaliana seeds expression of BnLPAT4-1 driven by napin indicated that BnLPAT4-1 can reduce the ratio of long-chain polyunsaturated fatty acid and saturated fatty acid, and specific improve the content of Eicosenoicacid.6. Through the technology of homology-based cloning one copy of LPAT5 full-length CDs sequence (1041bp) had been cloned from Xiangyou 15, and designated as BnLPAT5. Sequence analysis revealed that BnLPAT5 had the LPLAT_LCLAT1 like domain and belonged to the LPLAT superfamily.7. BnLPATS was a ubiquitous expression gene. Under the treatment of Nacl, PEG4000 and water logging, BnLPATS presented a model of’rapidly fall and then slowly ascent’and the minimum was at 3 h after treatment. As for the treatment of 6BA and ABA, BnLPATS presented a model of’slowly ascent and then slowly fall’and the minimum was at 12 h after treatment.8. Yeast expression of BnLPATS presented that its expression product have transferase activity. The ratio of unsaturaved fatty acid was 85.73%, it is 5.82 times of saturated one. The level of oleic acid and linoleic acid occupied 85% of the total fatty acid. BnLPAT5 is preference for oleic acid and linoleic acid, the order is linoleic acid>oleic acid.9. T3 Arabidopsis thaliana seeds expression of BnLPAT5 driven by napin indicated that BnLPAT5 can depress Arabidopsis thaliana LPAT selective for C22:1, C20:0, C20:2 and C20:3. Although BnLPAT5 in different plant showed different selective activity, it improved the ratio of long-chain low polyunsaturated fatty acid.