臺灣博碩士論文加值系統

論文摘要內容：
本實驗之目的為利用白蟻傳統分類外表形態測量，白蟻染色體數和染色體核型分析，RAPD標記、DNA定序，建立白蟻遺傳變異評估的方法，並運用染色體核型分析來做分類，藉由RAPD-PCR來建立白蟻彼此之親緣關係。將台灣家白蟻，黃肢散白蟻，黑翅土白蟻兵蟻，以頭部為中心進行傳統分類形態測量，結果顯示，傳統外表型態分類不易將台灣家白蟻和黑翅土白蟻之兵蟻分辨出來，但可由外表形態分辨出黃肢散白蟻兵蟻，與台灣家白蟻、黑翅土白蟻兵蟻之不同，外表形態無法用大顎長度來區分，但頭部其他特徵可用來鑑定區別。供試白蟻經染色體數檢查的結果顯示，台灣家白蟻之兵蟻、工蟻、有翅型生殖蟻，染色體數皆為2n＝66，屬二倍體，其他種如黑翅土白蟻工蟻染色體數為2n＝46，黃肢散白蟻工蟻染色體數為2n＝18，截頭堆沙白蟻工蟻染色體數為2n＝26，亦為二倍體。供試白蟻大部份是中部著絲點染色體(m)為主。核型分析結果顯示，台灣家白蟻之兵蟻、工蟻、有翅型生殖蟻，核型公式完全相同，皆是2n＝30m＋1sm＋2st，黑翅土白蟻工蟻為2n＝20m＋2sm＋1st，黃肢散白蟻工蟻為2n＝9m，截頭堆沙白蟻工蟻為2n＝11m＋2st，實驗結果顯示不同種白蟻其核型亦不同，可作為分類之依據。分析最長和最短染色體長度比值介於5.8-1.9之間。種間核型對稱係數分佈在47.4%-68.8%，顯示其核型對稱程度高。依據Stebbin核型分類，黃肢散白蟻工蟻核型分類1A，截頭堆沙白蟻工蟻核型分類2B，台灣家白蟻工蟻和黑翅土白蟻工蟻均為2C。
以30組隨機引子進行RAPD分析，其中有14組引子經PCR反應後，在電泳膠片上可產生多型性清楚DNA條帶，此14組引子產生的389個逢機增殖DNA片段，每一組引子平均可產生27.7個逢機增殖DNA片段，可以用來分辨不同的種。利用Jaccard (1908)和Nei & Li(1979) 所提出的相似度(similarity coefficient)公式來計算出不同種之間遺傳相似性及UPGMA方法所建立出來的親緣關係圖，分析結果顯示，截頭堆沙白蟻和供試的其他三種，黃肢散白蟻台灣家白蟻及黑翅土白蟻相距最遠，截頭堆沙白蟻為獨立一群，黃肢散白蟻，台灣家白蟻，及黑翅土白蟻為一群，同種白蟻工蟻和兵蟻間親緣關係，以台灣家白蟻間相似度最高達86.1%。因此這些引子可以作為種鑑定的標記以及種間遺傳變異性之評估。
以COⅠ、COⅡ、ITS2三區域基因做定序，結果顯示只有台灣家白蟻工蟻ITS2區域有產物產生，其產物長度約380個鹼基對。

Abstract
The objectives of this study were to establish a method to
assess the genetic variations of termites by measuring their
morphological characteristics used in the traditional taxonomy and
determining their number and karyotype of chromosomes as well as
analyzing the DNA sequence of Coptotermes formosanus Shiraki
by using RAPD markers. Also determined was the phylogenic
relationship among the three species of termites by using RAPD —
PCR. The results of the morphological measurements of the heads
of C. formosanus, Reticulitermes flaviceps (Oshima), and
Odontotermes formosanus (Shiraki) showed that it was difficult to
distinguish the soldier of C. formosanus from that of O. formosanus
on the basis their morphological differences. However, the soldier
of R. flaviceps was morphologically distinguishable from that of
C. formosanus and O. formosanus by other characteristics of their
heads instead of the length of their mandibles. The study of the
number of chromosomes showed that the soldier, worker and alate
castes of C. formosanus shared the same number of chromosomes
as diploid at 2n=66. The numbers of chromosomes for the worker
caste of O. formosanus, R. flaviceps, and Cryptotermes domesticus
were all diploids at 2n=46, 2n=18, and 2n=26, respectively.
Among the termites studied, the majority of their chromosomes
were metacentric (m). Results of the karyotype analyses showed
that all of the soldier, worker and alate castes of C. formosanus
shared the same karyotype as 2n=30m+1sm+2st. However, the
karyotypes of the soldier of O. formosanus, R. flaviceps, and
C. domesticus were 2n=20m+2sm+1st, 2n=9m, and 2n=11m+2st,
respectively. This showed that different species of termites had
different karyotypes; thus, karyotype analysis could be used as a
basis for termite classification. Analyses of the ratios of the
longest and shortest arms of a chromosome showed that they fell
within the range of 5.8 — 1.9. The study of the degree of
symmetry of karyotypes showed that the T.C. values for the four
species of termites studied fell within the range of 47.4% -- 68.8%,
indicating their high degree of symmetry in Karyotypes. Based on
the Stebbin’s karyotype classification, the karyotype for the worker
of R. flaviceps was classified as 1A, the worker of C. domesticus
classified as 2B, and the worker of both C. formosanus and
O. formosanus classified as 2C.
Results of the RAPD analysis showed that among the 30
random primers tested, 14 of them demonstrated clear, polymorphic
DNA bands on electrophoric gels. Using the 14 primers, a total of
389 DNA sections were randomly amplified, indicating an average
of 27.7 DNA sections amplified by each primer. This could be
used for distinguishing different termite species. Based on the
similarity coefficient proposed by Jaccard (1908) and Nei & Li
(1979), the genetic similarity among the termite species studied was
determined and the phylogenic relationship was also established by
using the UPGMA methods. Results of the study showed that C.
domesticus was determined as an independent group with the most
distant phylogenic relationship with three other species of termites,
including R. flaviceps, C. formosanus and O. formosanus.
R. flaviceps, C. formosanus and O. flaviceps were categorized in the
same group. Studies of the phylogenic relationship between the
worker and soldier castes showed that C. formosanus demonstrated
the highest degree of similarity between the two castes at 86.1%,
indicating that these primers could be used as the marker for the
species identification and for assessing genetic variations between
species.
Using the sequencing order of the three regions, COⅠ, COⅡ and ITS2, to determine the DNA sequences, the results showed that PCR product appeared only in the region of ITS2 with a length of 380bp.

目錄
中文摘要……………………………………………………….…Ⅰ
英文摘要……………………………………………………….…Ⅲ
誌謝…………………………………………………………….…Ⅴ
目錄…………………………………………………………….…Ⅵ
表次索引………………………………………………………….Ⅷ
圖次索引…………………………………………………….……Ⅸ
壹、前言…………………………………………………………...1
貳、前人研究…...…………………….…………………...………3
一、白蟻之分類地位及特徵………..………..………………...3
二、染色體數…………..…………………………….…………4
三、染色體核型分析………………………………….……..…4
四、種的鑑定之技術………..……………………………..…...7
參、材料與方法..……………………………………….……..…21
一、研究對象..……………………………………………...…21
二、白蟻傳統分類之形態測量……………….…………..…...21
三、白蟻染色體數調查及染色體形態………………….…….22
四、白蟻染色體核型分析的操作..……………………..…..…25
五、白蟻DNA RAPD-PCR分析…………..…………………26
(一)、DNA之萃取與純化…………………………..……...26
(二)、PCR與RAPD 產物電泳分析………………………27
(三)、逢機增殖DNA標記之引子篩選……………………28
(四)、核酸條帶資料分析…………………………………...28
(五)、白蟻DNA 之定序…………………………………...29
肆、結果…………………………………………………………...32
一、白蟻傳統分類形態測量…………………………...………32
二、白蟻染色體形態及數目的分布…………………………...33
三、白蟻染色體核型分析……………………………………...36
四、白蟻種類鑑定及親緣關係分析………………….………..38
(一)、隨機引子篩選……………………….………………...38
(二)、RAPD標記分析…...……………………….…………39
(三)、群叢分析……………………………………..………..43
(四)、DNA之定序……...…………………………………...45
伍、討論………...…………………………………………………46
陸、結論…………………………………………………………...54
柒、參考文獻……………………..…………………………….…55
作者簡介………………………………………………………...109
表次索引
表一、不同DNA的分子量所需的適宜循環次數………..…. 68
表二、DNA片段的PCR擴增………………………………...69
表三、按對稱到不對稱的核型分類…...………………………70
表四、家白蟻、黃肢散白蟻、黑翅土白蟻兵蟻之測量比較
..…………………………………………………….…..71
表五、家白蟻、黃肢散白蟻、黑翅土白蟻兵蟻之測量比較
..………………………………………………………...72
表六、家白蟻、黃肢散白蟻、黑翅土白蟻、截頭堆砂白蟻之工
蟻染色體數…………………...………………………..73
表七、家白蟻Coptotermes formosanus染色體數……………73
表八、家白蟻、黑翅土白蟻、黃肢散白蟻、截頭堆砂白蟻染色
體參數…………………...……………………………..74
表九、家白蟻、黑翅土白蟻、黃肢散白蟻、截頭堆砂白蟻的核
型特徵………………………………...………………..75
表十、14組Operon 引子其序列GC含量比率及增幅DNA
條帶數……………………………...…………………..76
表十一、利用Jaccard方法分析4種白蟻RAPD條帶計算
出不同種間的相似性………………….…………….77
表十二、14組引子之矩陣分析表…………..……...….……....78
圖次索引
圖一、家白蟻Coptotermes formosanus (Shiraki)有翅型成蟲….83
圖二、家白蟻Coptotermes formosanus (Shiraki)…………….....84
圖三、家白蟻Coptotermes formosanus (Shiraki)大顎……….....85
圖四、黑翅土白蟻Odontotermes formosanus (Shiraki)………...86
圖五、黃肢散白蟻Reticulitermes flaviceps (Oshima)………......87
圖六、截頭堆砂白蟻Crytotermes domesticus (Oshima)工蟻…..88
圖七、家白蟻Coptotermes formosanus工蟻染色體形態……...89
圖八、家白蟻Coptotermes formosanus兵蟻染色體形態……...90
圖九、家白蟻Coptotermes formosanus有翅型雄蟲染色體形
態...….………….…………………………..……….…….91
圖十、黑翅土白蟻Odontotermes formosanus工蟻染色體形
態. .……………………….…………………....…….…....92
圖十一、黃肢散白蟻Reticulitermes flaviceps工蟻染色體形
態.....………………...………………...………………..93
圖十二、截頭堆砂白蟻Crytotermes domesticus工蟻染色體
形態……………… .…………………………………...94
圖十三、家白蟻工蟻核型分析圖…………………………...…...95
圖十四、家白蟻兵蟻核型分析圖………………………………..96
圖十五、家白蟻有翅型雄蟲核型分析圖………………...……...97
圖十六、黑翅土白蟻工蟻核型分析圖…………………...……...98
圖十七、黃肢散白蟻工蟻核型分析圖………..……...……….....99
圖十八、截頭堆砂白蟻工蟻核型分析圖…………………...…100
圖十九、白蟻染色體數分析圖………………………………....101
圖二十、家白蟻染色體數分析圖……………………………...102
圖二十一、四種白蟻利用AP08 primer標記分析電泳膠片
圖譜………………..……………………………….103
圖二十二、四種白蟻利用OP04 prim標記分析電泳膠片圖
譜………………..………………………………….104圖二十三、4種白蟻間的親緣關係圖…………………………105
圖二十四、台灣家白蟻DNA以COl、COll、ITS2三特定
基因區域之六組引子進行PCR擴增的結果…….106
圖二十五、台灣家白蟻DNA以引子ITS2擴增的產物以
Hhal酵素切割的多態性…….………..…...……....107
圖二十六、白蟻ITS2基因部份片段序列圖…………………..108

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