| 摘要: | 台灣處於熱帶及亞熱帶地區，夏季高溫是青花菜栽培不易、產量不高的原因之一，青花菜受高溫傷害的程度與品種、生長期、器官、溫度、高溫逆境期長短有關。高溫逆境易引起植物蛋白質變性、氨毒害、膜傷害、光合作用下降、呼吸作用升高造成饑餓傷害等。本研究欲瞭解青花菜耐熱‘欣樺1208’與不耐熱‘優秀’二品種於不同溫度(25, 30, 35, 40℃)不同處理(0, 1, 3, 6, 12, 48 及72小時)下，植株生長、外部形態及葉片膨壓、電解質滲漏、抗氧化系統之差異與變化，評估酵素活性可否成為蔬菜品質與產量之生理指標，並將此酵素選殖出來。
以actin為internal control，利用real-time PCR分析在高溫逆境下抗氧化酵素mRNA之相對表現量。利用RACE法擴增其抗氧化酵素基因cDNA full length末端，經選殖入載體並定序後，至NCBI網站比對此cDNA full length序列，以確定此段基因。
結果顯示高溫造成青花菜產生外部形態的變化，葉片出現下垂、枯萎、萎縮、捲曲等症狀。而葉片膨壓、電解質滲漏無法明顯區別出耐熱與不耐熱品種。‘優秀’ 之CAT活性於熱逆境下增加較多，‘欣樺1208’APX活性於熱逆境下增加較多。
mRNA表現量‘優秀’於40℃處理6小時後，APX及CAT表現量皆大幅增加。‘欣樺1208’APX於35℃及40℃處理24小時後有所增加，40℃處理6小時後CAT表現量皆大幅增加。經RACE擴增出SOD、APX和CAT序列，將定序結果整理，送至NCBI blastn比對後，與阿拉伯芥SOD序列有93%、阿拉伯芥APX序列有81%、芥菜(Brassica juncea) CAT序列有99%以上的相似度，推論RACE擴增出之片段分別為SOD、APX和CAT cDNA序列。 本研究已將青花菜之CAT 及ubiquiitin全長ORF序列選殖出來。
Taiwan is located in the tropical and subtropical regions. Production of broccoli is limited to a large extent by climates with high temperatures. The growth and development of broccolis are strongly influenced by heat stress that can be initiated by oxidative stress. To cope with this stress, plants have developed a series of defense mechanisms and detoxification systems. The objectives of our work were to study the changes of antioxidant system in broccoli under 25, 30, 35 and 40℃ for 0, 1, 3, 6, 12, 48, and 72 hours, and identify any antioxidant system linked to heat-tolerance traits under heat stress. Heat-tolerant variety “SF1208 “ and heat-susceptibility variety “Green Magic“ were used to measure the antioxidant enzymes, turgor pressure and electrolyte leakage under the growth chamber. The response of target gene expressions and regulations under the stress were determined using real-time PCR. A full-length target cDNA was amplified using RACE technique followed by cloning it into the vector for sequencing, and comparing it with any known sequences in the NCBI-blastn.
The result shows that the increased catalase and ascorbate peroxidase activity provides the broccoli plants with increased tolerance. The turgor pressure and electrolyte leakage were not able to significantly identify the differences between genotypes. Both of the APX and CAT gene expressions were not well consistent to their activities. The sequences of SOD, APX and CAT were 93% and 81% identical to A. thaliana , as well as 99% to B. juncea, respectively. We have clones both catalase and ubiquiitin full length cDNA sequences (open reading frame) in broccoli. |
