Cloning And Expression Analysis Of The Key Enzymes In Fatty Acid Metabolism Of Peanut(Arachis Hypogaea L.)

Peanut (Arachis hypogaea L.) is a main oil crop in our country and the ultimate production occupies the first place in the world, its quality improvement has great significance for economic development of our country. When applying traditional breeding technology, there exist many problems such as long breeding cycle, limitation to improve special constituents and difficultation to integrate agronomic characters. Gene engineering has provided an effective approach for crop variety hereditary improvement because it could shorten the breeding cycle and increase the breeding efficiency. Consequently, improving fatty acid compositon of peanut by using gene engineering technique has important theoretical and pratical signficicance.In this stuty, in order to study the novel genes of peanut, a cDNA library from seedlings was constructed. Total RNA was extracted from E12peanut seedlings and mRNA was purified. Double strand cDNA was synthesized by SMART method, the dscDNA fragments were ligated into the pBluescript Ⅱ SK vector. The recombinant plasmids were transformed into the E. coli, a cDNA library of peanut seedlings was successfully constructed. The titer of the cDNA library was estimated as1.1×106cfu/mL; the percentage of recombination was as high as93.4%. PCR results showed that the inserts varied from0.75to2.0kb. Large-scale sequencing of cDNA was carried out to generate numbers of high-quality expressed sequence tag (EST) sequences. After sequence assembling and annotation,4074unigenes were generated.3897unigenes showed significant similarity to known proteins.Two ESTs of peanut PEPC (HS096-h02and HS092-d05) which are from the cDNA library of peanut seedling were cloned using RACE-technique. The sequences of peanut PEPC were designated as AhPEPC1(Accession Number:EU391629) and AhPEPC2(Accession Number:FJ222240), respectively. Additionally, we designed degenerate primers to clone three other peanut PEPC genes according to conserved amino acid sequences of PEPCs reported. The cDNAs of the three PEPC genes were obtained by RACE, designated as AhPEPC3(Accession Number:FJ222826), AhPEPC4(Accession Number:FJ222827), AhPEPC5(Accession Number: FJ222828). The obtained cDNA of AhPEPCI-5from peanut consists of2,907bp,2,901bp,2,901bp,2,910bp,3,111bp, which encodes968,966,966,969,1036amino acids, respectively. Real-time PCR analysis revealed that AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, AhPEPC5were expressed constitutively in four various tissues (root, stem, leaf and seed) of normal-oil variety and high-oil variety, but they were expressed in different pattern. The expression levels of AhPEPC1, AhPEPC3, AhPEPC4and AhPEPC5, except AhPEPC2, in normal variety were higher than in high-oil variety.Based on the EST sequence of peanut KASII gene (HS137_H08) and SAD gene (HS148_D02) obtained from the cDNA library, the cDNA sequences containing the complete CDS of KAS II gene and SAD gene were cloned by using RACE-technique respectively and the two cDNA sequences were designated as AhKASII (Accession Number:FJ358425) and AhSAD (Accession Number: FJ230310) respectively. AhKAS II consists of1867bp, encoding404amino acids and AhSAD consists of1499bp, encoding406amino acids. Expression analysis of KAS II gene and SAD gene in various tissues (root, stem, leaf and seed) from different peanut varieties were performed by real-time PCR technique respectively. Analysis showed that the expression levels of genes in various tissues were both distinctly different. The expression level of KAS II gene was highest in the seeds and the expression level of SAD gene in seeds and leave was distinctly higher than in other two tisses. That indicated both of the genes may play an important role in the seed growth and development. In addition, the expression levels of the two genes also differ in different varieties.By using bioinformatics method, the full-length cDNAs of△12fatty acid desaturase gene were isolated from peanut (Arachis hypogaea L.) genotypes with normal and high ratio of oleic to linoleic acid, which were designated as AhFAD2B and AhFAD2B’, respectively. Sequence alignment of their coding regions revealed that an extra A was inserted at the position+442bp of AhFAD2B’sequence of high-oleic-acid genotypes, which resulted in the shift of open reading frame, the premature termination of encoded region and a truncated protein AhFAD2B’, with the loss of one histidine box involved in metal ion complex required for the reduction of oxygen. Expression analysis showed that the expression of△12fatty acid desaturase gene in high-oleic-acid genotype was slightly lower than that in normal genotype. The enzyme activity experiment of yeast (Saccharomyces cerevisiae) cell transformed with AhFAD2B or AhFAD2B’proved that only AhFAD2B gene product showed significant△12fatty acid desaturase activity, but AhFAD2B’gene product did not. These results suggested that the change of AhFAD2B’ gene sequence resulted in lower activity or deactivation of△12fatty acid desaturase in high-oleic-acid genotype.