摘要
小麦是世界上最重要的农作物,也是我国仅次于水稻的主要的粮食作物。小麦花药培养的研究与利用是大幅度提高小麦育种效率的重要途径,但花药培养过程中愈伤组织诱导率及成苗率较低,则一直是花药培养技术发展的瓶颈。为了更好地利用小麦花药培养技术为农业发展服务,应加强对小麦花药培养力的基础性研究。
大量研究表明,小麦花药离体培养的最佳接种时期是小孢子发育在单核中晚期,这一时期的花药培养出愈率最高。而目前,前人的研究都是通过花药培养对比试验,统计出愈率等方法得出的结果,未能从蛋白质水平上解释其原因。有鉴于此,本实验以探索小麦花药培养在小孢子单核中晚期出愈率较高的原因,探求小麦花药发育过程中的蛋白质代谢机理,以期找到这一时期与小麦花药培养力有关的蛋白。
本研究选用经过小麦花药培养选育而成的陕农138品种为试验材料,采用蛋白质组学技术,对其小孢子发育在单核早期,单核中晚期和双核期三个时期的全蛋白质组进行了系统深入的研究。
主要研究结果与讨论如下：
1.利用SDS-PAGE电泳对其小孢子发育在单核早期,单核中晚期和双核期三个时期的全蛋白电泳谱带进行了分析。结果表明：小孢子发育到双核期时出现2条明显的条带或表达丰度大的条带,其分子量分别为50.7kDa,48.2kDa,而这两条带分别在单核早期和单核中晚期单独出现。
2.利用双向电泳技术对其小孢子发育在单核中晚期和双核期两个时期的全蛋白电泳图谱进行了分析,结果表明：(1)在等电点(pI)4-7之间均可识别约450个以上清晰的蛋白质点,检测到差异点26个,其中单核中晚期11个,双核期15个。在单核中晚期11个差异点中,5个在小孢子单核中晚期表达而发育到双核期时消失,6个在双核期表达量明显减弱。在双核期15个差异点中,5个在小孢子单核中晚期消失而发育到双核期时得到重新表达,10个在双核期表达量明显增强。(2)两时期在双向电泳和单向电泳中表达的蛋白质按MW分布情况十分相似,并且蛋白质点差异显著,更近一步证明了单向电泳结果的可靠性。
3.选取了14个差异表达的蛋白质点,通过基质辅助激光解吸分离飞行时间质谱进行肽指纹图谱分析(MALDI-TOF-MS),并利用Mascot软件在NCBInr数据库搜索,鉴定出了11个蛋白质点,另3个蛋白质点未得到有意义的鉴定,11个蛋白质点分别被鉴定为UDP-葡萄糖焦磷酸化酶、叶绿素抗体结合蛋白、pentatricopeptide重复蛋白PPR566-6、半胱氨酸蛋白酶抑制剂、泛素、2个蛋白质点被鉴定为S-腺苷-L-半胱氨酸水解酶、2个假定蛋白及2个放养增强蛋白。鉴定的蛋白质点其功能涉及到物质能量,糖代谢,蛋白质降解,细胞防卫等代谢过程,小麦花药发育的发生很可能与这些蛋白质的代谢体系相关。
Wheat is the most important crop in the world, which is also the major food crop in China. The research and utilization of wheat anther culture was an important way to improve the efficiency of wheat breeding, However, the induction rate of callus and seeding rate were very low in the process of anther culture, which was the bottleneck in the development of anther culture. the basic study on the anther culture ability of wheat should be strengthen, In order to service agricultural development by making better use of wheat anther culture technology.
The anther culture ability of wheat is the anther callus in the process of anther culture and the differentiation rate of green plantlet, which is affected easily by many factors, such as the genotypes of material, the developmental stage of anther microspore, media, culture conditions and so on. The developmental stage of anther microspore is very important to the induction rate of callus of anther culture in these factors. Many results showed that the mid and late uninucleate pollen was the best vaccination period in anther culture in vitro of wheat, in which the induction rate of callus of anther culture was the highest. At present, the induction rate of callus was calculated by comparative test of anther culture in the previous studies, which the reason could not be explained, so the reason that the induction rate of callus of anther culture was the highest in the mid and late uninucleate pollen and the mechanism of protein metabolism in the developmental stage of anther were explored in this paper, in order to find the protein that had the relation with the anther culture ability of wheat in this stage.
In this paper, the whole proteome at the early uninucleate pollen period, mid and late unimucleate pollen period and binucleate pollen period of microspore development was systematic studied by proteomics technology with the Shaannong138 as the material.
The main results and discusses were as follows:
1. The total protein bands at the early uninucleate pollen period, mid and late unimucleate pollen period and binucleate pollen period of microspore development were analyzed by SDS-PAGE electrophoresis in this paper. The result showed that two protein bands appeared at the binucleate pollen period of the microspore development, whose molecular weight of 50.7kDa,48.2kDa, and these two bands appeared alone at the early and mid-late uninucleate pollen period respectively.
2. The total protein bands at the mid and late unimucleate pollen period and binucleate pollen period of microspore development were analyzed by two-dimensional electrophoresis technology in this paper. The result showed that:(1) There were about 450 clear protein spots could be detected between the isoelectric point (pI)4 and (pI)7, in which there were 26 different protein spots. There were 11 different protein spots in the mid and late unimucleate pollen period, in which there were 5 spots disappeared and 6 spots significantly reduced from the mid and late unimucleate pollen period to binucleate pollen period. And there were 15 different protein spots in the binucleate pollen period, in which there were 5 spots re-expressed and 10 spots significantly enhanced in this stage. (2)The proteins that expressed in two-dimensional and one-way electrophoresis during the two stages were very similar according to the distribution of MW, and the difference of protein spots was extremely significant, which proved the reliability of one-way electrophoresis results.
3. The peptide fingerprinting (MALDI-TOF-MS) of 14 different protein spots were analyzed by matrix-assisted laser desorption time of flight mass spectrometry and using Mascot to search in the NCBInr database, which identified 11 protein spots, however, there were 3 protein spots could not be identified.1 protein spot was identified as UDP-glucose-1-phosphate uridylyltransferase, chlorophyll a-b binding protein, pentatricopeptide repeat protein PPR566-6, cysteine proteinase inhibitor A and ubiquitin; 2 protein spots were identified as S-adenosyl-L-homocysteine hydrolase, two hypothetical protein and two stocking-increasing protein. The functions of proteins identified were related to metabolic process of material energy, carbohydrate metabolism, protein degradation and cell defense, which were likely associated with the anther development of wheat.
引文
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