Investigation Of The Characteristics Of Floral Organ In Male Sterile Pepper And Cloning Of CaCP26 In Pepper And Its Analyse

The male sterile materials of pepper(HW200A, HW201A, HW202A, HW203A, HW204A) and their corresponding maintainer lines(HW200B, HW201B, HW202B, HW203B, HW204B) were provided by College of Horticulture of Northwest A & F University. Firstly I mainly compared six combinations of floral parts and made analysis. Secondly, I chose three combinations and observed the the microstructure of microspore development. According to the result of observation with light microscope, the combination named HW204A and HW204B was further observed under TEM(transmission electron microscope). I determined the pollen vitality of this combination and the number of pollen in an anther. Then morphological studies of pollen of pepper under SEM(scanning electron microscope) are observed. In addition, a positive different TDF(transcription derived fragment) was obtained from buds of the fertile individuals and the sterile individuals of genetic male sterile pepper named HW58 with cDNA-AFLP technique.The main results were obtained as follows:1. Observing and comparing the six different floral organs, the anthers of male sterile lines are smaller than that of maintainer lines, however, the filaments of the male sterile lines are longer than that of maintainer lines.2. Observing and analyzing the microspore developments of three combinations of male sterile lines and their corresponding maintainer lines named HW200A,HW201A,HW204A and HW200B,HW201B,HW204B with light microscope, two different types of the abortion occurred:(1)In HW200A and HW201A lines, pollen tetrads were all formed, but as the tapetal cells greatly inflated, broke down, and then sticked to the pollen tetrads. It finally could not form mononuclear microspore;(2)In HW204A line, the mononuclear microspore successfully formed, but could not form mature pollen grain.3. After the stage of mononuclear microspore, the tapetal cells broke down, sticked to the mononuclear microspore, then the mononuclear microspore broke down itself and finally resulted in abortion in HW204 under TEM.4. The determination of the vitality of pollen and the number of pollen in an anther: no pollen in male sterile line named HW204 with no vitality; 22625±2017.11 pollen grain in maintainer line named HW204B with 92.41%±2.63%.5 Based on the TDF, the cDNA which is 865bp was acquired by electronic cloned method. It was verified further in buds of the fertile individuals of genetic male sterile pepper named HW58 with RT-PCR technolgy. The sequence cloned in pepper had a high identity up to 99.61% with the sequence obtained from electronic clone. The result of Blast N demonstrated that the gene shared 90% homology to the CP26 of tobacco. So we named this gene as CaCP26(GenBank accession number GQ999612). The structure, the composition and character of amino acids of CaCP26 gene sequence were analysed by bioinformatics software. The gene was 871bp in length, including a 864bp clear open reading frame which codes 287 amino acid. The results were that its relative molecular weight is 30710.47 Da, isoelectric point was 5.087, a kind of hydrophobic protein. It had three transmembrane helics, without a signal peptide. The secondary structure of CaCP26 was mainly composed of random coil. Swiss-Model software predicted a tertiary structure including 65aa-285aa. A molecular evolution tree showed that CP26 of pepper had high genetic relationship with tobacco.6. The semi-quantitative RT-PCR was employed to analyse the expression of CaCP26 in buds of sterile individuals and fertile individuals in uninucleate stage, in which the fertile individuals exhibited higher relative expression than the sterile individuals.