臺灣博碩士論文加值系統

我們先前已從甘藷老化葉片中分離一SPCP3全長cDNA，能轉譯出一可能的含granulin半胱胺酸蛋白酶。葉片老化是葉子生長發育過程的最後一個階段而且已被視為是計畫性細胞凋亡（programmed cell death）的一種形式 。在植物PCD過程中主要參與的酵素有papain-type 半胱胺酸蛋白酶和Caspase-like 蛋白酶。最近一新奇的位於C端含有granulin的半胱胺酸蛋白酶形式已被了解且從植物多種不同情形下所誘導的PCD過程中被分離出來。Granulin是一古老的蛋白家族，在動物中參與調控細胞生長與刺激細胞分化。在植物中，C端含有一類似granulin區域的半胱胺酸蛋白酶的生理角色和功能目前尚不清楚，但報導推測植物中含granulin之半胱胺酸蛋白酶的角色可能與植物生長發育和逆境下所誘導之計畫性細胞凋亡的過程有關。本研究以扣減雜交的方式與重組載體技術，獲得分別含有不同部分SPCP3基因的重組載體pBI121。以農桿菌經由floret dip 方法產生基因轉殖阿拉伯芥。在確認含有全長SPCP3基因且在mRNA與蛋白質階層中皆有表現之T1轉殖植株的外觀形態分析顯示，轉殖T1植株與對照組相比有較早開花的現象。果莢成熟後的長度也較短，且果莢黃化的程度也較快。基於這些研究的結果，認為甘藷SPCP3是一功能性的老化相關基因，且似乎直接扮演與老化有關的角色。

We have previously isolated a full-length SPCP3 cDNA, which encodes a putative granulin-containing cysteine protease from sweet potato senescent leaves. Leaf senescence is the final stage of leaf development and has been considered as a type of programmed cell death. In plants the main enzymes involved in this process are papain-type cysteine proteases and Caspase-like proteases. Recently a novel type of cysteine proteases containing C-terminal granulin-like domains were identified and isolated from various plants under different conditions that induce PCD. Granulins are a family of evolutionarily ancient proteins that are involved in regulating cell growth and in stimulating cell division in animals. The physiological role and function of plant novel cysteine proteases containing C-terminal granulin-like domains were not clear. However, several studies concerning its association with programmed cell death during plant development and under stress conditions were described. In this study, we obtained recombinant pBI121 vectors containing different portions of SPCP3 inserts by deletion and recombinant vector technology. Transgenic Arabidopsis plants harboring different SPCP3 portions described above will be produced with Agrobacterium-mediated transformation via floral dip. Morphological analysis in T1 transgenic plants containing full length SPCP3 which that have been charactered expression in mRNA and protein level, which that displayed T1 transgenic plants flowered earlier, the length of siliques is shorter and the degree of senescence of siliques is more evident. These results support that sweet potato SPCP3 is a functional senescence-associated gene and likely plays a role directly in association with senescence.

內容目次
頁次
第一章 前言
ㄧ、葉片的老化………………………………………………………… 1
二、計畫性細胞凋亡（PCD )……………………………………………2
三、C端含granulin之半胱胺酸蛋白酶………………………………… 4
四、Granulin 相關之研究 ………………………………………………5
五、含Granulin區域之半胱胺酸蛋白酶的相關研究 …………………6
六、研究基因功能性之基因轉殖植株系統…………………………… 9
七、研究目的 ………………………………………………………… 11
第二章 材料與方法
Ⅰ材料………………………………………………………………… 13
1) 選殖於pGEM-T easy 載體之甘藷老化葉片中所分離之梭基端含
有Granulin區域的半胱胺酸蛋白酶SPCP3之全 cDNA ( GenBank
Accession no. AF259983 ) …………………………………………13
2) 載體 pGEM-T easy ……………………………………………… 13
3) 載體pBI121 ……………………………………………………… 13
4) 大腸桿菌DH5α（E. coli DH5α）…………………………………14
III
5) 農桿菌LBA4404（Agrobacterium tumefaciens LBA4404） ………14
6) 抗體 ………………………………………………………………14
Ⅱ方法
一、重組載體的構築……………………………………………………15
A.重組載體pGEM-T easy與大腸桿菌DH5α勝任細胞的轉
形作用（transformation）: ………………………………………15
B. 小量純化質體DNA（使用QIAGEN的QIAprep Spin Miniprep 套
組) : …………………………………………………………… 17
C. 聚合酶鏈鎖反應：………………………………………………18
D. 瓊酯糖凝膠中DNA片段的萃取( QIAGEN的 QIAquick Gel
Extration 套組 ) ：……………………………………………… 19
E. 沈澱反應（precipitation）：………………………………………20
F. 黏合反應（pGEM-T east vector system I，Promega）:……………21
G. 大腸桿菌DH5 α 勝任細胞(Invitrogen) 的轉形作用與藍白篩
選：………………………………………………………………22
H. 載體pGEM-T easy選殖基因片段之DNA序列分析：………… 25
I. 大量製備載體pBI121： …………………………………………25
1) 在營養培養基中擴增載體pBI121
IV
2) 鹼裂解法
3) 使用QIAprep Spin Miniprep 套組純化質體DNA
J. 構築重組載體pBI121：………………………………………… 28
二、農桿菌的電穿孔轉形作用…………………………………………33
A. 農桿菌生化檢測……………………………………………… 33
B. 農桿菌勝任細胞的製備與電穿孔轉形作用………………… 34
三、阿拉伯芥的花序浸泡轉形作用產生轉殖植株……………………35
四、轉殖阿拉伯芥T0植株的篩選、種植及基因組PCR………………36
A. 轉殖阿拉伯芥T0植株的篩選與種植…………………………36
B. 基因組PCR …………………………………………………… 39
五、轉殖阿拉伯芥T1植株的定性分析 ……………………………… 42
A. 轉殖阿拉伯芥T1植株的篩選 ……………………………… 42
B. χ2- test 遺傳分離法則符合度測驗………………………… 42
C. Total RNA之分離與RT-PCR ………………………………… 43
D. 蛋白質的之電泳分析與蛋白質膠體墨點雜交分析 ……… 46
六、 轉殖阿拉伯芥T1植株生長發育過程與外觀形態之觀察………53
第三章 結果
一、 含不同部分半胱胺酸蛋白分解酶基因SPCP3 ( 全長SPCP3基因、
V
SPCP3基因減除C端類似Granulin區域與只含C端類似Granulin區域之
cDNA片段 ) 之pBI121重組載體的構築： …………………………55
二、農桿菌之基因轉殖：………………………………………………58
三、轉殖阿拉伯芥T0植株之篩選與Genomic PCR確認： ………………59
四、轉殖阿拉伯芥T1植株之定性分析：………………………………61
五、轉殖阿拉伯芥T1植株外觀型態之比較：…………………………62
第四章 討論
一、轉殖阿拉伯芥植株之轉殖效率：…………………………………65
二、轉殖阿拉伯芥植株含有SPCP3基因：……………………………65
三、SPCP3表現會促進轉殖T1植株提早開花：………………………67
四、轉殖T1植株花序中SPCP3表現會影響果莢發育：………………69
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