‘无籽’瓯柑小孢子母细胞减数分裂特性基因RAD51和MS1的表达差异分析

Expression of microsporocyte meiosis with special genes RAD51 and MS1 in Citrus suavissima ‘Seedless'

‘无籽’瓯柑Citrus suavissima‘Seedless’是野生型瓯柑Citrus suavissima的芽变,其花粉完全败育,败育起始于小孢子母细胞减数分裂形成四分体时期。研究‘无籽’瓯柑小孢子母细胞减数分裂异常的分子机制,有利于深入认识果树芽变的发生机制。随机选择"无籽"瓯柑和瓯柑各3株,根据花蕾直径采集花粉母细胞时期(Ⅰ),四分体时期(Ⅱ),单核花粉粒时期(Ⅲ),双核花粉粒时期(Ⅳ)及花粉粒成熟期(Ⅴ)共5个时期的花蕾并分离花药,利用实时荧光定量聚合酶链式反应(q RT-PCR)分析减数分裂特性基因RAD51和MS1的相对表达量,3次重复。通过SPSS 16.0的最小显著差法(LSD)在0.01水平上进行各样品间的相对表达量差异分析。结果表明:RAD51和MS1基因的表达高峰均集中在第Ⅰ时期和第Ⅱ时期,且此时花药中的相对表达量均极显著(P<0.01)高于花蕾。第Ⅰ时期花药中,‘无籽’瓯柑RAD51的相对表达量为瓯柑的3.7倍,MS1可达300倍;但同时期花蕾中,RAD51的相对表达量仅1.1倍,MS1为140.0倍。‘无籽’瓯柑RAD51基因在第Ⅰ时期和第Ⅱ时期的相对表达量极显著(P<0.01)高于瓯柑,说明‘无籽’瓯柑小孢子母细胞和四分体时期的细胞内均存在DNA受损现象;‘无籽’瓯柑MS1基因在第Ⅰ时期的相对表达量极显著(P<0.01)高于瓯柑,但第Ⅱ时期极显著(P<0.01)低于瓯柑,推测‘无籽’瓯柑败育花粉的形成与MS1基因表达紊乱引起油脂分泌、转运异常密切相关。

To obtain the relative expression of two meiosis special genes- RAD51 and MS1 （Male Sterity 1 ） ,Citrus suavissima ‘Se ed less’ , a bud variation of its wild type Ougan （Citrus suavissima） with completely aborted pollens that appear abnormally from the tetrad stage, were studied through the molecular mechanism of abnormal meiosis. According to flower bud diameter, five stages of flower buds and anther -pre-meiotic （PM, Ⅰ）, tetrad （T,Ⅱ） , single-celled （SC, Ⅲ）, dual-core （DC, Ⅲ ） , and mature pollen （MP, Ⅳ ）-- were collected and each was an composite sample from three trees. An experiment in real-time quantitative polymerase chain reaction （qRT-PCR） was conducted by three replications in each sample and the method of least significant difference （LSD） with SPSS 16.0 was used to test the expression differences between samples under the level of P 〈0.01. Results showed that expression of RAD51 at Stage I of the anther and the flower buds for C. suavissima #Seedless＇ was significantly higher （P〈 0.01 ） than Ougan. At Stage I of the anther, the relative expression of RAD51 in C. suavissima ‘Seedless’ was 3.7 times higher than in Ougan with MSI reaching about 300 times higher. However, at Stage I of the flower buds, ＂-$51 was only 1.1 times higher and MSI was about 140 times higher. The significantly higher expression of RAD51 at Stage I indicated that a phenomenon of DNA damage might exist in the microsporocyte and tetrad periods of C. suavissima ‘ Seedless ’ ; for gene MS1, significantly higher expression of C. suavissima ‘ Seedless ’ for Stage I also meant that the aborted pollens of C. suavissima ‘ Seedless ’ had a relationship with the abnormal secretion and transfer of oil lipids that were influ-enced by disordered expression of MS1. [Ch, 6 fig. 1 tab. 24 ref.]