【作者】 黎云祥；

【导师】 陈放；

【作者基本信息】 四川大学 ， 植物学， 2002， 博士

【摘要】 珙桐生活史中较独特的发育事件是其较小的头状花序下形成的2～3个大型苞片。一般认为，苞片是叶片的变态或自叶片演化而来。然而自1869年珙桐被发现以来，未见关于珙桐苞片在该物种进化生物学、生殖生态学及其生活史中的作用和地位的报道。该物种是研究植物苞片发育的分子机理及探讨苞片与叶片发育的相互关系的理想材料，也是探讨苞片在植物生殖生物学及生殖生态学中作用和功能的理想材料。 本研究利用新近发展的抑制性差减杂交技术(SSH)成功构建了珙桐幼嫩苞片和叶片差减文库。共分析了237个克隆的插入片段，其大小介与100到2000bp之间。用正向扣除产物和反向扣除产物作探针分别与237个克隆的插入片段PCR产物杂交，结果表明有167个克隆代表了只在苞片中表达或在苞片中表达量增加的基因。用地高辛标记的珙桐苞片、叶片和根的cDNA做探针，与差减杂交所获得克隆的PCR产物的杂交结果表明，有少数基因在这些器官中都是大量表达，而大多数在这些器官里的表达量均很少，除苞片外，一般均检测不到，有部分基因在苞片中优势表达。 随机挑选了16个差异表达的克隆进行序列测定，经与GenBank数据库相关数据比较分析，发现有2个新的cDNA片段(ETS)，有12序列与GenBank中已知蛋白或推测蛋白质(putative protein)序列有高低不同的同源性，它们参与基因的表达调控、植物的次生代谢、信号传导、抗逆、应激及防御反应等细胞生命活动过程。另外还有2个序列与数据库中已存在的未知蛋白质同源。这些 四川大学博士学位论文 基因片段在抑制性差减杂交前后的量均有较大的变化，即差减扣除后被大大富 集了。 分析本试验获得的烘桐苞片差异表达的有限几个部分或全长序列的cDNA 发现：＊）处于生长发育初期的苞片中有一些较为独特的基因表达。如PIB4基 因编码的葡萄糖昔酶、PIAS基因编码的植物脂肪转移蛋白、PZD4基因编码的 一种富含脯氨酸的蛋白都有参与植物的抗逆、防御或应激反应的功能八2）幼嫩 苞片中有互为桔抗作用的基因同期表达。如编码脱落酸诱导或成熟过程中表达 蛋白的PIF4基因与编码植物生长素响应蛋白的．PIHll基因都同期在苞片中优 势表达。 通过抑制性差减杂交的到克隆PIAS，反向NOrthern杂交表明它在玖桐苞片 中优势表达，即在苞片中的表达水平远远超过在处于同时期的叶片中的表达水 平。RTPCR结果证明它在苞片中强烈表达而在处于同时期的叶片中几乎检测不 到其表达。对得到的该片段分析表明，它是一种脂肪转移蛋白（LTP）基因的部分 序列。采用 5’－端 RACE技术获得了该基因的全长 CDNA序列＊ N。其推测 的氨基酸序列与GenBank中注册的LTP蛋白前体的同源性达63％，且具有植物 Lh蛋白共同特征，如8个保守的半肌氨酸位点，较高的等电点oI＝＝8．9似及缺 少色氨酸等。推测的氨基酸序列具有两个显著的跨膜螺旋：第一个螺旋位于第5 个（Gly和第35个仰）氨基酸之间，第二个螺旋位于第28（Alg和第46队eul个氨 基酸之间。推测的最可能的信号肽切割位点位于第28（Alal和29（Alal个氨基酸之 间。椎测的成熟肽共有92个氨基酸，分子量9．ZkDa。上述所有证据表明，我们 克隆到一个烘桐苞片优势表达的脂肪转移蛋白基因NsLTP。构建T PI AS和 poE30的重组质粒，转化大肠杆菌M15，IPTG诱导表达，Westhe杂交证实PIAS 在大肠杆菌中得到了表达。更多还原

【Abstract】 A unique developmental aspect involved in the life cycle of Davidia involucrata Baill. is the formation of two or three large-sized attractive white bracts surrounding a relatively small-sized capitulum. It has long been believed that plant bract is one kind of metamorphism of or evolved directly from leaf. Nonetheless nothing has been done on its role and function in the evolutionary and reproductive biology and in the life cycle of this species, saying nothing of its developmental mechanism, since the species was found from West China in 1869. This species is one of perfect materials to examine the developmental mechanism of plant bract, to explore the developmental relationship between leaf and bract, as well as to study the role and essential function of bract on plant reproductive biology and ecology.In virtue of the technique of suppression subtractive hybridization, we had successfully constructed a subtractive library of D. involucrata sprouting bract by taking homochronous leaf as driver in this study. Differential screening for the library revealed that, 167 of 237 clones randomly selected from the library are positive and represent genes that were exclusively or intensively expressed in bract, which contain inserts ranging from 100 to 2000 bp in length. Moreover, except a few clones were expressed predominantly in bract, absolute majority of subtraclive clones were expressed at low level in all checked organs, i.e., bract, leaf and root.Among sequenced 16 positive clones randomly selected, two represent novel expression tag sequences, two are homologous to two unknown proteins in GenBank; the rest are homologous to known or putative proteins or enzymes with definite functions by searching the GenBank through Blast program, which are involved in various life activities of cell such as regulation of gene expression, plant secondary metabolism, signal transduction, adversity resistance, stress response and defense reaction. Significant changes of quantities of these gene fragments were observedbefore and after SSH, which indicated they were enriched after SSH.Sequence analyses for those limited amount of inserts indicated that, (1) A few particular gene populations are expressed in D. involucrata sprouting bract, e.g., gene population encoding proteins that involve in plant defensive reactions and adversity resistance, such as P1A5 gene encoding a non-specific lipid-transfer protein, P1B4 gene encoding a raucaffricine-O-p-D- glucosidase and P2D4, encoding a proline-rich protein; (2) Gene populations with antagonistic effects occurred simultaneously in sprouting bract, e.g., P1F4 gene encoding an ABA stress ripening protein versus P1 H11 gene encoding an auxin-responsive protein.Reverse Northern hybridization indicated that P1A5 gene was over-expressed in bract compared with that in leaf. This expression pattern was further conformed by RT-PCR. Sequence analyses revealed that it is a partial sequence of a lipid transfer protein gene. A complete cDNA of 1047 bp was obtained by means of 5’-RACE (5’-Rapid Amplification of cDNA End) techniques using gene specific primer P1A5-1. The deduced amino acid sequence of P1A5 was most homologous to the lipid transfer protein 3 precursor isolated from upland cotton, the lipid transfer protein SDi-9 isolated from common sunflower, and the nonspecific lipid-transfer protein precursor allergen pru av3 isolated from sweet cherry. It also had common features of plant nsLTPs (non-specific lipid transfer proteins) such as eight conserved cysteine positions, high isoelectric points (8.9) and lack of tryptophans. The deduced amino acid sequence had two strong transmembrane helices, i.e., the first from position 5 (Gly) to position 35 (Val) and the second from position 28 (Ala) to position 46 (Leu). The cleavage site of putative signal peptide was predicted to occur between 28(Ala) and 29(Ala) thus the putative mature form of the protein composed of 92 amino acids with a molecular weight of 9.2 kDa. All these provide compelling evidence that the P1A5 clone b更多还原