Cloning And Expression Analysis Of Gene Related To Fertility In Male-Sterile Wheat Lines

Male sterility is one of the major characteristics to be used in heterosis utilization of crops. Male sterile wheat lines are valuable for heterosis study and utilization. To inverstigate the molecular basis of male fertility conversion in male sterile wheat lines, based on the previous study, MADS-box transcription factor and Ras-GTPase activating protein genes were cloned in male-sterile lines and their maintainers of three types of male sterile wheat lines and two thermo-sensitive male sreile wheat lines. Then, sequence analysis between the two thermo-sensitive male sreile wheat lines and three types of male-sterile lines and their maintainers were conducted. The expression profiles of the two genes in those materials were further analyzed via semi-quantitative RT-PCR. The main results were as follows:1. An EST (Genebank accession number: 36925702) highly similar to MADS-box transcription factor gene was selected from the sterile SSH-cDNA library of YS type thermo-sensitive male sterile wheat line A3017 and used as the probe to search the dbEST. A pair of primers was designed based on the aligned sequence of highly homological EST sequence. Then the RT-PCR fragment amplified from the spikes of male-sterile lines and their maintainers of three types of male sterile wheat lines were cloned and sequenced, 6 cDNA sequences with about 660bp were obtained. BLAST analysis at NCBI website and Prediction of conserved domain showed these sequences were the target gene. Multiple alignment analysis showed that these sequences have small differences of single nucleotide. Clustering analysis showed that the sequence of male-sterile lines gathered in one cluster and the sequence of maintainers gathered in another cluster. It suggests that the expression of MADS-box transcription factor gene between male-sterile lines and their maintainers were determined by their nucleotide sequences.Expressions of MADS-box transcription factor gene between three types of male-sterile lines and their maintainers were analysed via semi-quantitative RT-PCR. The expression of this gene in male sterile spikes was higher than that in fertile spikes. It suggests that the expression of this gene is related to the fertility conversion of male-sterile lines.2. An EST (Genebank accession number:DY543200) highly similar to Ras-GTPase activating protein gene was selected from the fertile SSH-cDNA library of YS type thermo-sensitive male sterile wheat line A3017 and used as the probe to search the dbEST. A pair of primers was designed based on the aligned sequence of highly homological EST sequence. Then the RT-PCR fragment amplified from the male-sterile lines and their maintainers spikes of three types of male sterile wheat lines were cloned and sequenced, 6 cDNA sequences with about 1300bp were obtained. BLAST analysis in NCBI website and Prediction of conserved domain showed these sequences were the target gene. Multiple alignment analysis showed that these sequences have multiple single nucleotide differences.Expressions of Ras-GTPase activating protein gene between three types of male-sterile lines and their maintainers were revealed via semi-quantitative RT-PCR. The expression of this gene in maintainer spikes was higher than that in male-sterile spikes. It suggests that the expression of this gene is related to the fertility conversion of male-sterile wheat lines.3. Used the EST (Genebank accession number:DY543200) that highly similar to Ras-GTPase activating protein gene as probe to designed a pair of primers, 8 DNA sequences with about 2700bp were obtained in the genome DNA of male-sterile lines and their maintainers of three types of male sterile wheat lines and two thermo-sensitive male sreile wheat lines. BLAST analysis in NCBI website and Prediction of conserved domain showed these sequences were the target gene. Multiple alignment analysis showed that these sequences have small differences of single nucleotides. Alignment Analysis on exon profile between the cDNA sequence of male-sterile lines and their maintainers of three types of male sterile wheat lines and their DNA sequences showed that all of these DNA sequences have 8 exons, the core areas and equivalent cDNA sequences were the same. But there are multiple mismatches of exon between DNA sequence and cDNA sequence. We postulate that this gene take placed small quantity of base mutation in the edit process, and lead to fertility conversion. Clustering analysis showed that the DNA sequence of male-sterile lines gathered in one cluster and the sequence of maintainers gathered in another cluster. In addition, the two thermo-sensitive male sreile wheat lines were gathered in one subcluster. It suggests that the expression of this gene are different between male-sterile and fertility conditions.