甘蓝型油菜温敏核不育系SP2S小孢子释放过程相关基因的表达分析

doi:10.7668/hbnxb.2014.02.007

摘要/Abstract

摘要： 用组织化学染色观察甘蓝型油菜温敏核不育系SP2S小孢子释放过程中胼胝质累积、降解情况,并用定量PCR检测花蕾不同发育阶段的小孢子释放相关基因表达情况。结果表明:苯胺蓝染色荧光检测发现甘蓝型油菜雄性不育系SP2S四分体及小孢子被大量胼胝质包裹,而且降解困难,壁物质降解延迟使得小孢子粘连在一起;对胼胝质酶基因BnMSR66进行扩增及测序,所得片段长度为445 bp,SP2F和SP2S材料的cDNA和DNA序列完全相同。包含一个全长为390 bp的开放阅读框,编码130个氨基酸;对花蕾不同发育时期参与四分体外周壁上纤维素降解的基因BnQRT1、BnQRT2、BnQRT3及胼胝质降解相关基因BnA6和BnMSR66进行实时荧光PCR定量分析,这些基因在不育株中表达量比可育株中大幅度提高,胼胝质过多而且延迟降解趋势与荧光染色观察结果一致。表明绒毡层变形及功能异常导致小孢子释放有关酶基因表达异常与小孢子败育有关。

关键词: 甘蓝型油菜, 温敏雄性不育, 细胞学, 胼胝质, 荧光定量PCR

Abstract: Thermo-sensitive genic male sterile line of Brassica napus plays an important role in two-line breeding. In this study, the new thermo-sensitive genic male sterile line of Brassica napus and its near isogenic line SP2F were used. The degradation of callose in SP2F and SP2S anthers was observed by aniline blue staining. The total RNA was extracted from different developmental stages of bud of lengths of 0-1,1-2,2-3,3-4 mm and reversely transcribed to cDNA. Amplification, sequencing and Real-time PCR analysis of genes involved callose dis- integration in microspore separation between SP2F and SP2S. The results indicated that: The callose of the SP2S was not degraded at tetrad stage, which made the microspores adhesion with each other and then pollen aborted. The cloned gene BnMSR66 belongs to plant β-1,3-glucanase genes and share a high similarity with other plants. The full length of BnMSR66 is 445 bp, and the ORF is 390 bp, encoding 130 amino acids, no difference between SP2S and SP2F. The expression of five genes, including BnMSR66 and BnA6 involved in callose dissolution, and three QRT genes (BnQRT1, BnQRT2, BnQRT3) control microspores separation after meiosis and produce mature pollen tet- rads, at four stages (PMC and meiosis stage, tetrad to the microspore released stage, uninucleate microspore, and the bicellular microspores stage, buds of 0-1, 1-2, 2-3, 3-4 mm length) were quantitatived by Real-time PCR, showed that all the five genes were significantly up-regulated in SP2S than in SP2F. Results of both cytological ob- servation and differential expression analysis exhibited that deformation and dysfunction of the tapetum lead to ab- normal expressing of the genes involved in microspore separation and microspore abortion.

Key words: Brassica napus L., Thermo-sensitive male sterility, Cytology, Callose, Real-time fluorescent quanti- tative PCR

中图分类号:

胡玉梅, 徐献锋, 于澄宇, 郭英芬, 葛娟. 甘蓝型油菜温敏核不育系SP2S小孢子释放过程相关基因的表达分析[J]. 华北农学报, 2014, 29(2): 38-44. doi: 10.7668/hbnxb.2014.02.007.

HU Yu-mei, XU Xian-feng, YU Cheng-yu, GUO Ying-fen, GE Juan. Cytological Observation and Expression Analysis of Genes Involved in Anther Tetrad Releasing of a Thermo-sensitive Male Sterile Line SP2S in Brassica napus L.[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2014, 29(2): 38-44. doi: 10.7668/hbnxb.2014.02.007.

http://www.hbnxb.net/CN/Y2014/V29/I2/38

参考文献

[1] 于澄宇. 甘蓝型油菜授粉控制系统研究现状与前景[J]. 西北农林科技大学学报,2011,39(9):1-8.
[2] 席代汶,陈卫江,宁祖良, 等. 甘蓝型油菜温敏核不育系湘油 91S 的选育[J]. 湖南农业科学,1994(4):17-18.
[3] 孙晓敏, 李玮, 冯志峰, 等. 油菜生态型不育系 373S小孢子败育的电镜观察[J]. 中国农学通报, 2011, 27(7):123-132.
[4] 葛娟, 郭英芬, 于澄宇, 等. 甘蓝型油菜光、温敏雄性不育系 Huiyou50S 花粉败育的细胞学观察[J]. 作物学报,2012,38(3):541-548.
[5] 杨光圣, 瞿波, 傅廷栋, 等. 甘蓝型油菜 3 种类型雄性不育系花药败育的细胞学观察[J]. 华中农业大学学报,1999,18(6):520-523.
[6] Dun X L, Zhou Z F, Xia S Q, et al. BnaC. Tic40, a plastidinner membrane translocon originating from Brassica oleracea, is essential for tapetal function and microspore develop-ment in Brassica napus[J]. Plant J,2011,68(3):532 -545.
[7] 郭英芬, 葛娟, 于澄宇, 等. 新型甘蓝型油菜温敏核不育系 SP2S 的花药发育解剖学观察[J]. 植物生理学报,2012,48(3):282-288.
[8] Li L W,Xiu Y X,Deng F H, et al. Abnormal vacuolizationof the tapetum during the tetrad stage is associated with malesterility in the recessive genic male sterile Brassica napus L.Line 9012A[J]. Plant Biol,2010,53(2):121 -133.
[9] Zhou Z, Dun X, Xia S, et al. BnMs3 is required for tapetaldifferentiation and degradation, microspore separation, andpollen-wall biosynthesis in Brassica napus[J]. J Exp Bot,2012,63(5),2041-2058.
[10] Rhee S Y, Somerville C R. Tetrad pollen formation inquartet mutants of Arabidopsis thaliana is associatedwith persistence of pectic polysaccharides of the pollenmother cell wall[J]. Plant J,1998,15(1):79-88.
[11] Heslop-Harrison J, Mackenzie A. Autoradiography of sol-uble (2-14C) -thymidine derivatives during meiosis andmicrosporogenesis in Lilium anthers[J]. Cell Sci,1967,2:387-400.
[12] Waterkeyn L, Beinfait A. On a possible function of thecallosic special wall in Ipomoea purpurea (L. ) Roth[J]. Grana,1970,10(1):13-20
[13] Peirson B N, Owen H A, Feldmann K A, et al. Character-ization of three male-sterile mutants of Arabidopsis thaliana exhibiting alterations in meiosis[J]. Sex Plant Re-production,1996,9(1):1-16.
[14] Frankel R, Izhar S, Nitsan J, et al. Timing of callase ac-tivity and cytoplasmic male sterility in Petunia[J]. Bio-chemical Genetics,1969,3(5):451-455.
[15] Stieglitz H. Role of β-1, 3-glucanase in postmeiotic mi-crospore release[J]. Developmental Biol,1977,57(1 ):87-97.
[16] Wood P J, Fulcher R. G. Specific interaction of anilineblue with (1-3 ) -β-D-glucan [J]. Carbohydrate Poly-mers,1984,4(6):49-72.
[17] 高菊芳, 杨太为. 荧光显微术在鉴别拟南芥花粉壁发育相关基因功能中的应用[J]. 上海师范大学学报:自然科学版,2010,39(6):616-622.
[18] Hird D L, Worrall D, Hodge R, et al. The anther-specificprotein encoded by the Brassica napus and Arabidopsisthaliana A6 gene displays similarity to β-1,3-glucanases[J]. Plant J,1993,4(6):1023-1033.
[19] Yang S L,Xiea L F,Mao H Z,et al. Tapetum determinatis required for cell specialization in the Arabidopsis anther[J]. Plant Cell,2003,15(12):2792-2804.
[20] Yamaguchi T, Nakayama K, Hayashi T, et al. Molecularcloning and characterization of a novel β-1, 3-glucanasegene from rice[J]. Biosci Biotechnol, Biochem,2002,66(6):1403-1406.
[21] Preuss D,Rhee S Y,Davis R W,et al. Tetrad analysis pos-sible in Arabidopsis with mutation of the QUARTET (QRT)genes[J]. Science,1994,264(5164):1458 -1518.
[22] Rhee S Y, Osborne E, Poindexter P D, et al. Microsporeseparation in the quartet 3 mutants of Arabidopsis im-paired by a defect in a developmentally regulated polyga-lacturonase required for pollen mother cell wall degrada-tion[J]. Plant Physiology,2003,133(3):1170-1180.
[23] 杨怡姝,孙晓娜,王小利,等. 实时荧光定量 PCR 技术的操作实践[J]. 实验室研究与探索,2011,30(7):15 -19.
[24] 胡瑞波, 范成明, 傅永福, 等. 植物实时荧光定量 PCR内参基因的选择 [J]. 中国农业科技导报, 2009, 11(6):30-36.

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