Bowman-Brik胰蛋白酶抑制剂在水稻叶片生长和种子萌发过程中的表达

Bowman-Birk胰蛋白酶抑制剂(BBTI)是一类富含半胱氨酸的丝氨酸蛋白酶抑制剂,广泛分布于豆科、禾本科和菊科植物中,在蛋白质存储和植物防御中具有重要作用。水稻(Oryza sativa L.ssp.indica)BBTI家族具有13个成员,其中BBTI-9和BBTI-10都为假基因。其余BBTI成员都存在数量不同的保守Bowman-Birk结构域。为了解BBTI的表达信息,本研究主要通过设计多肽序列的方法,制备了11个BBTI的多克隆抗体,利用Western blot技术检测其在叶片生长发育和种子萌发过程中的表达。结果表明BBTI在叶片生长过程中呈现不同的时空特异性表达,BBTI-1、-2、-11、-12和-13伴随叶片生长含量增加,BBTI-5、-6和-8在苗期表达含量高,而BBTI-3、-4、-7在苗早期1cm时表达量达到峰值,以修饰或多聚体的形式在叶片生长过程中表达。在种子萌发过程中发现BBTI-8在种子中表达量较高,萌发后2h降到最低,24h后一条分子量略小的降解带开始表达,其余抗体在种子萌发阶段未发现有明显的变化。本研究还统计了苗期叶片、分蘖期叶片和萌发期的种子的MPSStags数据,试图比较转录与蛋白质水平对应关系。本研究结果证明,BBTI蛋白质在水稻生长发育过程中存在差异表达,为深入了解其功能提供了重要线索。

Expression of Bowman-Birk Trypsin Inhibitor at Different Development Stages in Rice Leaves and Seed Germination

Bowman-Birk trypsin inhibitor (BBTI) is a type of cysteine-rich serine protease inhibitors, which is widely distributed in Leguminousaes, Gramineaes and Asteraceaes plants, and it plays an important role in protein storage and plant defense. There are 13 members of BBTI in rice genome, among which BBTI-9 and BBTI-10 are both pseudogenes, while others all include different amounts of Bowmen-Birk domains. To research the expression of BBTI proteins, 11 BBTI specific antibodies were generated mainly by the method of peptide sequence design and synthesis, and the expression patterns at leaf growth and seed germination stages of rice(Oryza sativa L. ssp. indica) were separately revealed by Western blot. The results indicated that BBTI showed specific temporal and spatial expression at various development stages in leaves. Expression level of BBTI-1, -2, -11, -12, and a-13 increased steadily with the growth of leaves, while that of BBTI-5, -6, and -8 reached the highest level at seedling stage. BBTI-3, -4, and -7 expressed most in 1cm at early seeding stage, presented expression in forms of modification and polymer during the leaf growth. Expression of The BBTI-8 protein was high in seeds and declined to the lowest level at 2 h after germination, and a degrading band of smaller molecular weight was expressed at 24 h. However, others were not found obvious changes in expression during seed germination. In attempt to compare the corresponding relationship of RNA and protein level, we collected statistics about MPSS tags of leaves at both seeding and tillering stages and seeds with germinating period. In sum, this experiment revealed differential expression patterns of BBTI proteins during growth and development stages in rice, providing important clues for a better understanding their functions.