植物性病原菌近年來分子生物學及結構生物學的快速發展, 加上眾多基因體的DNA定序陸續完成, 使得結構基因體學變成當今重要的研究課題。求得蛋白分子的三維結構有助於對此蛋白質分子生化功能之瞭解。因此結構基因體學之研究, 即強調對某個基因體眾多蛋白質三維結構的瞭解, 並進而探討其相互作用及功能。

Xanthomonas campestris為格蘭式陰性病原菌，為一頗俱學術性及應用性的菌種。在學術上1)它能分泌多種胞外蛋白, 己成為研究革蘭氏陰性菌蛋白分泌之模式系統; 2)它的轉錄單位多以單基因的方式為之, 與熟知的大腸菌之多單基因調節方式不同; 3)它不具 cyclic AMP（cAMP），但其多元調控蛋白（Clp，cAMP receptor protein-like protein）能夠協助執行調控之功能。在應用上為1)它可攻擊十字花科植物，使之感染黑腐病，因而造成農業之損失; 2)此病菌可生成Xanthan（為一多醣體），可用在眾多工業如紡織、造紙等。基於此病原菌在學術上及農業及工業應用上巨大的潛力，本追求卓越計畫擬從事此病原菌的結構基因體研究, 以期對此一病原菌做一更全面性的瞭解。首先基因定序組將利用shotgun法負責將Xanthomonas caanthomonas campestris (Xcc)含約五百萬對鹼基對的基因體加以定序，並提供含有將近一千個基因的clones，以供後續的生物資訊組將整個基因組作系統化的分析，以提供利用實驗決定蛋白結構的依構。接著蛋白表達組將利用生物資訊組所提供的基因註解，建立一有效的蛋白載体体系，以供蛋白質高度表達之用、並做蛋白純化及功能分析的工作。接下的實驗則為利用多維核磁共振及X-ray繞射分析法解出它們的三維結構，求得其結構與功能之關係。為了要克服分子量太大的蛋白質所造成T2太短而譜線太寬的缺點，我們擬用TROSY的技術來增加核磁共振光譜的解析度。同時為了能在較短時間內能收集較多的多維光譜, 以達到一年解出約十個左右蛋白質結構的進度, 我們亦擬用cryoprobe的硬体技術, 使訊號的強度增加三倍, 而數據收集的時間減少三倍以上。我們同時亦將利用自動化光譜判定（Autoassign）以輔助人工判讀，及自動化結構產生(AutoStructure)軟体來加速蛋白質三維結構的決定。我們亦將利用蛋白質体學及DNA微陣列研Clp的調控網, 以了解受Clp調控的基因及其彼此的互動。我們也擬利用SOS誘導的篩選方法, 將整個Xcc基困体中, 找出直接或間接造成DNA損害的因子。同時也將建構不同gene knockout的變体以研究未知基因的功能。此計劃的最終目的為提供一個含有結構和功能註解的Xanthomonas campestris結構基因庫。相信本計畫的完成，不僅在學術上有極重要的意義(可發現一些含有特殊折疊的蛋白質結構及功能及未知功能的基因及Xcc所俱特殊的生化調節等)，在工業上及農業上也對十字花科植物黑腐病的預防及Xanthan的應用，有相當大的助益。同時它對台灣在國際學術地位的提升及培養台灣急需的高級生物科技人才亦將有立竿見影的功效。

The flood of sequence information available from the various genome projects coupled with the recent advances in molecular and structural biology has led to the concept of structural genomics on a genome-wide scale. Determination of the three-dimensional structures of proteins is critical for understanding the biological basis of the protein molecules. In this context, ‘structural genomics’ is expected to pave way for understanding the intricate interactions among proteins in the whole organisms.

Xanthomonas campestris is a gram-negative bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. However, it also produces an exopolysaccharide called xanthan that is of great industrial importance. Owing to the immense economic impact and the simplicity of the genetic constitution (lacking introns) of this organism, we envisage, in the present proposal, to identify and characterize novel protein structures and functions in Xanthomonas campestris. Our genome sequencing team will first use shotgun approach to sequence the whole genome of Xanthomonas campestris that contains approximately five million base pairs to allow bioinformatics team to identify and annotate the genome sequence. Such annotation is necessary to serve as the ground for protein expression team (using PCR to amplify the genes or using the clones provided by genome sequencing team) to engineer efficient vectors for expressing gene products in high yield and to purify and analyze their functions. After samples are ready, the structure team will then use high resolution NMR methodology or X-ray diffraction methodology to determine the solution structures of novel target proteins. To circumvent the problem of short T₂ relaxation and broad linewidth in large proteins, we plan to employ a variety of Transverse Relaxation-Optimized Spectroscopy (TROSY) technique to optimize the resolution. We will further use cryoprobe to accelerate NMR data collection so that we can collect enough data in a short time to solve approximately ten protein structures in a year. We will also use autoassign program to help assign the multidimensional spectra that is the most time-consuming part in protein structural determination. Autostructure determination program will also be used to accelerate the whole process. In addition, we intend to obtain high precision structures of the target proteins using the now popular dipolar coupling restraints. Attempts would also be made to identify potential binding substrate(s) for the target proteins using the SAR by NMR approach. The final goal of this coordinated project is to obtain a structural database of Xanthomonas campestris containing all structural and function annotation. We believe that successful execution of this project will not only promote academic excellence (to find novel proteins with novel folds), but also serve industrial and agricultural need (to prevent black rot disease and to provide xanthan gum). It will promote the academic status of Taiwan and provide personnel Taiwan desperately need in the biotechnology sector to advance into the 21-century.