Expression Analysis Of Fertility Genes From Genic Male Sterility A/B Line And Isolation Of Genes Related To Microspore Development In Chinese Cabbage-pak-choi (Brassica Campestris L.ssp. Chinensis Makino)

Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino; syn. Brassica rapa L), originated from China, is one of the vegetable cultivars and possesses the largest cultivated area among all vegetable crops in China. Chinese cabbage-pak-choi has a long history of cultivation, and is affluent in resources of cultivars. Chinese cabbage-pak-choi is a typical allogamy plant and has redominant heterosis. Much attention has been_paid to research on the plant breeding and application of male sterility. Mechanism of male sterility is complex, which involved a set of structural and regulative genes in microspore development. The analysis of those genes associated with microspore development should contribute to elucidate the mechanism.We studied male sterility A/B lines of Chinese cabbage-pak-choi about their spatial and temporal expression characters at morphological, physiological and molecular levels. Differential bands from B line were isolated and four full-length cDNA sequences related to the development of microspore were cloned .The results were as follows:(1) Plants growth status, physiological and biochemical factors were compared between A line and B line of genie male sterile A/B line of Chinese cabbage-pak-choi. Results showed that anthers of sterile plants were small, and pollens were abnormal or even no pollen at all; during rosette stage the vegetative growth of the A line was consistent with the B line, but in the blooming stage, the growth_of the A line was remarkably higher than the B line. We deduced that the mainly possible cause of these results was that the floral buds of the male fertile plants need more energy to keep the higher level of cyanide-resistant respiration, because we found the cyanide-resistant respiration in the buds of the B line plants was significantly higher than the ones of the A line plants. There was no significant difference between the photosynthesis rates of leaves in different stages. IAA was absent but ZT was surplus in the buds of the sterile plants. The peroxidase isoenzyme bands of the middle-size and large alabstrums in the A line were more obvious than those_in the B line, but no apparent difference in little alabstrums and leaves. Two more obvious cytochrome oxidase isoenzyme bands existed in little and middle-size alabstrums in B line. However, there was no apparent different band of cytochrome oxidase isoenzyme of leaves and large alabstrums at different stages. Two specific bands in soluble proteins electrophoresis in the B line was found, but only one was found in the A line.(2) The study of plants regeneration from petiole explants of genie male sterile A/B line of Chinese cabbage-pak-choi was made. Results showed that petiole explants were surfacesterilized by rinsing in 75% ethanol for 30s followed by treated with 0.1%(w/v) aqueous mercuric chloride for 9 min was the best treatment; the concentration of 6-BA, NAA, and their ratio affected the inducing rate of adventitious shoot and root; the most suitable hormone combination to sterile explant was 2 mg/L BA and 0.2mg/L NAA, and inducing rate of adventitious shoot reached 75.0%; however, to the fertile explants, the combination was 2 mg/L BA, 0.1 mg/L NAA, whose adventitious shoot regeneration rate was 65.6%; the best root inducing medium comprised 0.1 mg/L NAA and 7.5 mg/L AgNC^.(3) Improving cDNA-AFLP technique and pjeamplified PCR conditions were_as follows: including 20 cycles and 2.3 mmol/L MgCl2, annealing at 53"C. Mg2+ concentration of selective amplification was also 2.3 mmol/L. cDNA-AFLP analysis revealed differential expression was showed in alabstrums and among leaves, floral stalk and alabstrums; however.no difference in rosette leaves, floral leaves and floral stalk leaves was found. Using three of the twenty-two pairs of primers, four differential bands were amplified in middle-size alabstrums and large alabstrums of the B line. With a pair of primers, expression abundance of a differential band in middle-size alabstrums of fertile line was...