| Eucommia ulmoides Oliv. is a single species and the genus of family Eucommiaceae, which is an important economic tree and precious traditional Chinese herbal medicine. E. ulmoides has strong adapt ability to the environment and widely distributes in 27 provinces lines between 27 and 42 degrees north latitude in China. The oil content of E. ulmoides kernels is about 30%, the seed oil is rich in α-linolenic acid and the content of α-linolenic acid can reach as high as 62%. The research shows that FAD3 is the key gene that directly catalyze the conversion of linoleic acid into α-linolenic acid. Therefore, it is important to research the FAD3 gene 0f E. ulmoides for studying the synthesis and efficiently accumulation of α-linolenic acid of molecular mechanisms in E. ulmoides kernel, oriented genetic improvement of E. ulmoides. We used the kernels of 70 and 160 days after flowering(short for DAF) in ‘HuaZhong No.6’ and ‘HuaZhong No.10’ as materials for transcriptome sequencing analysis respectively, and carried out a number of studies in the structure of FAD3 gene, gene expression, subcellular localization and function on that basis. The main results of the research were as follows:1. The transcriptomes of kernels of 70 and 160 DAF(days after flowering) in varieties ‘HuaZhong No.6’ and ‘HuaZhong No.10’ were sequenced by Illumina HiSeqTM 2000, a new generation of high-throughput sequencing technology. The results showed that a total of 72, 791,399 clean reads fragment including 14,702,548,161 bp(14.70 G) in sequence information were generated, and then de novo assembly generated a total of 96,469 unigenes with an average length of 690 bp, which contains 66.56 Mb in sequence information. Every index was high quality and reliability. Among them, 49,856 Unigenes accounted for 51.68 % of all-unigene were annotated by Blast searches. All 38,983 annotated unigenes according to GO category were divided into three categories(cellular components, molecular function and biological processes) of 56 branches by gene ontology; 14,796 annotated unigenes based on COG category were grouped into 25 functional categories; KEGG pathway analysis presented that 11,260 annotated unigenes were broadly divided into 117 classes according to its function. There were 9 621 complete SSR in the 96,469 unigenes, accounting for 84.14% of the total SSR. The complete SSR included 55 frequent motifs, and the highest repeat of complete SSR type was A/T(4,597), following by AG/CT(2,597)、AT/AT(439). This research was the first comprehensive transcriptome analysis for E. ulmoides kernels, providing an important foundation and reference value for further characterizing the functional genes involved in the process of seed development.2. The two FAD3 genes were cloned by using the RACE technology and transcriptome data, the information of the full-length cDNA, genome DNA sequence and promoters are as follows:(1) The full-length cDNA of EuFAD3-1 gene was 1,281 bp which encodes 426 amino acids with an estimated molecular weight of 49.18 kDa and isoelectricpoint of 7.43; the length of genome DNA sequence of EuFAD3-1 gene was 3,151 bp, containing eight introns and nine exons; the length of promoter of EuFAD3-1 gene was 2,038 bp;(2) The full-length cDNA of EuFAD3-2 gene was 1,347 bp which encodes 448 amino acids with an estimated molecular weight of 50.92 kDa and isoelectricpoint of 9.01; the length of genome DNA sequence of EuFAD3-2 gene was 1,347 bp, no introns; the length of promoter of EuFAD3-2 gene was 2,201 bp.3. The expression of EuFAD3 gene in different developmental stages of ‘Huazhong No.6’ and ‘Huazhong No.10’ kernels was studied by using realtime fluorescent quantitative PCR technology which used Actin as the referencegene:(1) The qPCR analysis showed that the expression trends of EuFAD3-1 gene in the ‘Huazhong No.6’ and ‘Huazhong No.10’ kernels were increased first and decreased later, and reached a maximum at 110 days and 150 days after flowering, respectively;(2) The expression trends of EuFAD3-2 gene in the ‘Huazhong No.6’ kernels increased first and decreased later, and then increased again, the maximum expression was 110 days after flowering; The expression trends of EuFAD3-2 gene in the ‘Huazhong No.10’ kernels decreased first before reached its maximal levels at 100 daysafter flowering and then decreased at last.4. According to the cloned cDNA sequence of two FAD3 genes, we designed specific primers, constructed the fusion expression vector contained genes of interest by utilizing pDNOR222.1 vector and the expression vector with YFP fluorescent protein(nYFP-CD3-686), transformed into Agrobacterium tumefaciens GV3101 by electroporation and injected into the leaves of wild small-leaved tobacco, then observed the subcellular localization of target genes by using the LSM 510 Meta confocal laser scanning microscope after co-localized with marker. The result showed that the two FAD3 gene of E. ulmoides located in the endoplasmic reticulum.5. The two plant over-expression vectors which were pCAMBIA1304-35s-EuFAD3-1 and pCAMBIA1304-35s-EuFAD3-2 were constructed by using recombinant DNA technology and transformed into Agrobacterium GV3101 by electroporation, tobacco leaves were infected by using leaf disc method and selected the positive plants with antibiotic. We got 30 and 36 positive transgenic tobacco plants respectively after the identification of positive transgenic tobacco plants by PCR and GUS assay which were using the genome DNA of positive transgenic tobacco plants as template. Determination of content and fatty acid composition of oil by using the efficient gas chromatography after selected nine tobacco strains seeds respectively, the result showed that compaired with the wild tobacco, the compound of α linolenic acid of tobacco seeds which were transferred into pCAMBIA1304-35s-EuFAD3-1 and pCAMBIA1304-35s-EuFAD3-2 gene was increased 4.27 % and 0.92 % respectively, but the compoud of Linoleic acid was decreased 3.86 % and 0.65 % respectively, other fatty acid components have no obvious change. The result showed that the compoud of α linolenic acid of over expression of pCAMBIA1304-35s-EuFAD3-1 gene in tobacco seeds is much higher than EuFAD3-2. |
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