Construction Of Ssh Library Related To Multi-ovary And Clone, Expression Analysis Of Correlation Gene In Wheat

Due to the unique characteristics of the floral organ and with multiple ovaries, the multi-ovary wheat can enhance the propagation coefficient and play a important role in the development of hybrid wheat. Previous research has focused on the development of floral organs, biochemical analysis of seed germination and molecular markers, as well as gene localization and genetic analysis. About the molecular mechanism of the formation of multi-ovary trait is needed to study at present. The NIL of multi-ovay wheat DuoⅡshowed the same genetic background except for the ovary character. Therefore, it was an excellent material for studying the formation mechanism of multi-ovary trait in wheat. In this study, the materials mentioned earlier were initially employed to construct two cDNA libraries of multi-ovary and mono-ovary trait by suppression subtractive hybridization (SSH). Through sequencing, alignment analyses and functional annotation of the expressed sequence tags (ESTs), the types of expressed genes and gene functions have been compared with the 2 libraries. The expression analysis (different materials, different stages and different organs) and clone of partial gene was researched. We hope to give some explanations to the molecular mechanism of the formation of multi-ovary trait. The results were listed as follows:1. The penetrance, maturing rate and percentage germination of the NIL of the multi-ovary wheat DuoⅡwas investigated and analyzed. At the same time, the phenomenon of 4~6 pistil in one floret was found during the process of investigation in the field. The penetrance was 77.2～95.3% in different multi-ovary lines and the average was 88.6%. The average of maturing rate of multiple seeds was 79.9%. The percentage germination of the seeds on main location of multi-ovary line and the seeds of mono-ovary line was no difference. The percentage germination of the seeds on additional location of multi-ovary line was lower than the seeds of mono-ovary line. The new mutation phenomenon of multi-ovary wheat was a result of carpelloid stamens (the stamens changed into multi-pistils) though analysis. The 4~6 pistil cannot be fertilized.2. The multi-ovary line and mono-ovary line of the NIL were employed to construct two cDNA libraries related to multi-ovary (Mu) and mono-ovary (Mo) by SSH. About 2536 clones were obtained from Mu library and there were 2383 positive clones, the recombination frequency was 93.97%. About 4543 clones were obtained from Mo library and there were 4359 positive clones, the recombination frequency was 95.95%. Random differentially expressed cDNA clones were screened by PCR (Nest primer or M13 primer) amplification in the two subtraction libraries. More than 85% of the clones contained an insert and while most of the cDNA inserts were 400~500 bp, the range of clones extended from more than 250 bp to less than 750 bp. Though extraction of plasmid and Enzyme digested of some positive clones, the inserted fragment mainly concentrated approximately 450bp. This results was similar to PCR.3. One hundred positive clones were randomly selected and sequenced from the Mu library and Mo library respectively. The average size of EST in Mu library was 378bp. Ninety EST sequences were obtained after removal of low-quality and repeated sequences. Sixty-five ESTs (72.22% of the 90 ESTs) had translated sequences homologous to proteins with a known function. Among the sixty-five ESTs, 27.69% of them were categorized into protein synthesis, whereas, 10.77% of them were involved in subcellular localization, 7.69% of them were related to energy and transcription respectively, 6.15% of them were related to metabolism, development and protein with binding function respectively, and a small proportion of ESTs was related to protein fate, signal transduction mechanism and cell defense. The average size of EST in Mo library was 339bp. Eighty-nine EST sequences were obtained after the removal of the repeated sequences. Sixty ESTs, accounting for 67.42%, had sequence similarity with known function proteins. Among the sixty EST sequences, 33.33% were unclassified protein. 11.67% of the annotated ESTs were associated with metabolism, 10% of them were related to the subcellular localization, and 6.67% of them were in relation to the energy, protein synthesis, protein fate and protein with binding function respectively. The proportion of different functional proteins was inconsistent between the two libraries. Proteins related to protein synthesis, transcription and development were found frequently in the Mu library. Some proteins associated with metabolism, protein fate and cellular transport were found frequently in the Mo library.4. The expression patterns of 12 genes from two library were analyzed by RT-PCR. The results showed that six genes from Mu library were up-regulated by a different extent in multi-ovary wheat. The expression differences of 40S ribosomal protein S6, snRNP G and ribosomal protein L10A were more significant than those of ribosomal protein S15A , 14-3-3 protein and 60s ribosomal protein L21. Different expression of putative CER1, chaperonin and putative prolyl 4-hydroxylase were not significantly different. In contrast, expression analysis of genes from the Mo library showed that UDP-D-glucuronate decarboxylase and ribosomal protein L17-1 were up-regulated, while the expression level of cysteine proteinase inhibitor showed no significant difference. Most genes'expression presented an up-regulated tendency at the 1~12mm development stages of young ear. The examined genes were abundantly expressed in different tissues and the quantity of expression for most of them was highest in stem shoot meristem. Study speculated that differential expression genes may be involved in the occurrence of multi-ovary trait.5. The EST highly similar to RPS15a and RPL21 gene from the SSH library was used as a query probe to blast the Genbank databases. Based on the assembled homologous cDNA sequence, both cDNA and DNA sequences encoding RPS15a and RPL21 were isolated and characterized by RT-PCR and PCR. The cDNA sequence of RPS15a in wheat (GenBank accession No.HM055513) was 413 bp in length and the open reading frame encoded a peptide of 130 amino acids. The DNA sequence of RPS15a (GenBank accession No.HM063421) was 642 bp in length, which contained three extrons and two introns. The cDNA sequence of RPL21 (GenBank accession No.HM138480) was 521bp in length and the open reading frame encoded a peptide of 164 amino acids. The homology of amino acids of RPL21 was not conserved between the plants and animals. The DNA sequence of RPL21 (GenBank accession No.HM138481) was 1600bp in length, which contained two extrons and one intron. According to expression analysis, the expression quantity of the RPS15a and RPL21 gene in 2～4 mm ears of multi-ovary line was higher than that in mono-ovary line. They are may be related to the occurrence of multi-ovary trait. Their function was needed to more study.6. The EST highly similar to Cab gene from the SSH library was used as a query probe to blast the Genbank databases. Based on the assembled homologous cDNA sequence, the cDNA sequence encoding Cab were isolated and characterized by RT-PCR. The cDNA sequence of Cab (GenBank accession No.HM138480) was 862bp in length and the open reading frame encoded a peptide of 266 amino acids. The cDNA sequence of Cab and the EST highly similar to Cab gene from the SSH library was assembled and then the cDNA sequence of Cab became 924bp in length. The expression quantity of Cab gene in 2~3 and 3~4mm ears of mono-ovary line was lower than that in multi-ovary line. The expression of Cab gene in 2~3mm ears was lower than that in other stages in 1~6mm ears. The expression quantity of Cab gene was highest in young leaves and no detectable in young roots.