杨树木质素合成酶PAL基因的克隆、鉴定及其表达载体的构建
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摘要
杨树是我国重要的造林树种，由于它速生、木材是纸浆优良的原材料而引起人们的广泛关注。我国杨树资源非常丰富，如何定向改良我国现有的杨树，培育工业用材林新品种，提高木材的利用率，解决我国木材短缺的现状是林木遗传育种工作者的首要任务。本文主要在杨树木质素生物合成酶基因苯丙氨酸氨解酶（PAL）的克隆、测序、鉴定以及其表达载体的构建方面开展了一些工作。
PAL是木质素生物合成途径中涉及到的第一个酶，它的丰度直接影响着生物体内木质素及许多酚类物质的代谢，因此对PAL进行基因操作具有重要的实际意义。我们在国内首次利用RT-PCR从杨树次生木质部提取的mRNA中扩增并克隆出木质部特异表达的PAL基因片段，并且利用pBI 121和pBin438构建了其表达载体，并做了初步的转化研究。我们取得的初步结果有：
1．通过引物分析软件，首先设计合成了用于PAL扩增的正向及反向引物，通过对杨树叶片DNA的特异PCR扩增，我们能够扩增出大小约600bp左右的单一DNA片段条带，这和我们所设计的片段大小基本相当，从而证明我们所设计的引物是正确的、实验条件是可行的。
2．我们在国内首次利用RT-PCR技术从杨树次生木质部提取的mRNA中扩增出一个大小为565bp的木质素合成特异表达的PAL基因片段。 将其克隆到pGEM-T Easy载体上，获得了重组质粒。通过对该重组质粒进行了限制性内切酶酶切、PCR扩增以及序列测序分析，证明我们插入到载体上的片段即是我们所扩增出来的DNA片段；通过和Osakabe Y等（1995）在EMBL核酸序列库中发表的白杨杂种（Populus kitakamiensis）PAL的cDNA序列（D30656）比较，二者的前400个碱基几乎完全相同，其同源性高达95％以上，从而证明我们所扩增出来的DNA片段即为PAL基因片段的一部分。我们将此质粒定名为pGEM-T -PAL质粒。
3．通过对pGEM-T-PAL质粒和载体pGEM-7Zf(+)进行EcoR I单酶切，分别得到PAL片段和pGEM-7Zf(+)片段。利用CIAP对pGEM-7Zf(+)的EcoR I单酶切片段进行脱磷酸化处理后，将二者进行连接，构建了PAL的中间载体——pGEM-7Zf(+)-PAL质粒，经测序分析PAL片段是以正向插入到pGEM-7Zf(+)酶切位点的。
4．同时对pGEM-7Zf(+)-PAL质粒和pBI 121载体进行Xba I和BamH I双酶切；对pGEM-7Zf(+)-PAL质粒和双元载体pBin438也进行Xba I和BamH I双酶切，然后分别进行连接，构建了表达载体质粒pBI121-PAL和pBin438 PAL。
5．将表达载体pBI121-PAL和pBin438 PAL转化农杆菌LBA4404，经对从农杆菌中提取的质粒分别进行特异引物扩增，能扩增出600bp的目的片段，证明表达载体已经转入了农杆菌。然后利用农杆菌转染杨树叶片和嫩茎，期望能得到转基因植株。
6．作为该项目总体目标的前一部分，我们基本完成了载体的构建工作。下一步就是转基因植株的组培、分子生物学鉴定以及转基因植株的木质素测定工作，希望能达到木质素含量或木质素组成上发生改变的植株，为社会创造出巨大的经济效益和社会效益，为我国杨木的材性改良做出贡献。
Poplar is one of the most important tree species in China, due to its fast growing and easy pulping. How to improve its timer utilization efficiency remains a problem for tree breeders. This study focuses on reducing its lignin content by manipulation of lignin biosynthesis enzyme gene.
PAL (Phenylalanine Ammonia-Lyase, EC 4.3.1.5) is the first enzyme ahead the multibranched phenylpropanoid metabolism including the lignin synthesis in higher plants. In the present study, we have amplified the PAL gene fragment using mRNA extracted from the developing xylem, cloned it in a T-vector, identified by sequencing and further constructed in the plant expression vectors. The major results of this dissertation are as follows:
We have designed the forward and reverse primer and used them to amplify the PAL gene from poplar (Populus euramericana 74/76) developing xylem mRNA. The amplified fragment is about 600 base pairs as we expected. This indicated our designation of the primers is adequate.
The fragment was cloned into pGEM-T vector. The insert was identified by restriction enzyme, PCR amplification and sequencing. The sequence of the amplified DNA fragment was 565 base pairs. Alignment with the Populus kitakamiensis cDNA sequence of PAL retrieved from EMBL nucleotide acid database (accession number D30656) showed that the first 400 base pairs in both sequences were almost identical. Therefore we believed that the fragment was amplified from the PAL gene.
To introduce proper restriction sites for subsequent cloning into the plantexpression vector, both the pGEM-7Zf (+) vector and the pGEM-T-PAL were cut by EcoR I to release the PAL fragment and the pGEM-7Zf (+) fragment. The two fragment were joined together to produce a new plasmid pGEM-7Zf (+)-PAL.
The pGEM-7Zf (+)-PAL, the binary vector pBI121 and pBin438 were cut by Xba I and BamH I to release the PAL fragment and the large fragments of pBI121 and pBin438. After ligation of the PAL fragment with pBI121 or pBin438 large fragment, we obtained two expressional vectors, named pBI121-PAL and pBin438-PAL respectively. The vectors were further transformed into Agrobacterium tumefeciens LBA4404.
The two constructions with sense and anti-sense direction of the PAL gene will be further introduced into poplar using Agrobacterium-mediated transformation to obtain the transgenic poplar plants. Both the PAL activity and the lignin content of the transgenic trees will be measured to study the effect on expression both sense and anti-sense PAL RNAs.
PAL是木质素生物合成途径中涉及到的第一个酶，它的丰度直接影响着生物体内木质素及许多酚类物质的代谢，因此对PAL进行基因操作具有重要的实际意义。我们在国内首次利用RT-PCR从杨树次生木质部提取的mRNA中扩增并克隆出木质部特异表达的PAL基因片段，并且利用pBI 121和pBin438构建了其表达载体，并做了初步的转化研究。我们取得的初步结果有：
1．通过引物分析软件，首先设计合成了用于PAL扩增的正向及反向引物，通过对杨树叶片DNA的特异PCR扩增，我们能够扩增出大小约600bp左右的单一DNA片段条带，这和我们所设计的片段大小基本相当，从而证明我们所设计的引物是正确的、实验条件是可行的。
2．我们在国内首次利用RT-PCR技术从杨树次生木质部提取的mRNA中扩增出一个大小为565bp的木质素合成特异表达的PAL基因片段。 将其克隆到pGEM-T Easy载体上，获得了重组质粒。通过对该重组质粒进行了限制性内切酶酶切、PCR扩增以及序列测序分析，证明我们插入到载体上的片段即是我们所扩增出来的DNA片段；通过和Osakabe Y等（1995）在EMBL
3．通过对pGEM-T-PAL质粒和载体pGEM-7Zf(+)进行EcoR I单酶切，分别得到PAL片段和pGEM-7Zf(+)片段。利用CIAP对pGEM-7Zf(+)的EcoR I单酶切片段进行脱磷酸化处理后，将二者进行连接，构建了PAL的中间载体——pGEM-7Zf(+)-PAL质粒，经测序分析PAL片段是以正向插入到pGEM-7Zf(+)酶切位点的。
4．同时对pGEM-7Zf(+)-PAL质粒和pBI 121载体进行Xba I和BamH I双酶切；对pGEM-7Zf(+)-PAL质粒和双元载体pBin438也进行Xba I和BamH I双酶切，然后分别进行连接，构建了表达载体质粒pBI121-PAL和pBin438 PAL。
5．将表达载体pBI121-PAL和pBin438 PAL转化农杆菌LBA4404，经对从农杆菌中提取的质粒分别进行特异引物扩增，能扩增出600bp的目的片段，证明表达载体已经转入了农杆菌。然后利用农杆菌转染杨树叶片和嫩茎，期望能得到转基因植株。
6．作为该项目总体目标的前一部分，我们基本完成了载体的构建工作。下一步就是转基因植株的组培、分子生物学鉴定以及转基因植株的木质素测定工作，希望能达到木质素含量或木质素组成上发生改变的植株，为社会创造出巨大的经济效益和社会效益，为我国杨木的材性改良做出贡献。
Poplar is one of the most important tree species in China, due to its fast growing and easy pulping. How to improve its timer utilization efficiency remains a problem for tree breeders. This study focuses on reducing its lignin content by manipulation of lignin biosynthesis enzyme gene.
PAL (Phenylalanine Ammonia-Lyase, EC 4.3.1.5) is the first enzyme ahead the multibranched phenylpropanoid metabolism including the lignin synthesis in higher plants. In the present study, we have amplified the PAL gene fragment using mRNA extracted from the developing xylem, cloned it in a T-vector, identified by sequencing and further constructed in the plant expression vectors. The major results of this dissertation are as follows:
We have designed the forward and reverse primer and used them to amplify the PAL gene from poplar (Populus euramericana 74/76) developing xylem mRNA. The amplified fragment is about 600 base pairs as we expected. This indicated our designation of the primers is adequate.
The fragment was cloned into pGEM-T vector. The insert was identified by restriction enzyme, PCR amplification and sequencing. The sequence of the amplified DNA fragment was 565 base pairs. Alignment with the Populus kitakamiensis cDNA sequence of PAL retrieved from EMBL nucleotide acid database (accession number D30656) showed that the first 400 base pairs in both sequences were almost identical. Therefore we believed that the fragment was amplified from the PAL gene.
To introduce proper restriction sites for subsequent cloning into the plant
The pGEM-7Zf (+)-PAL, the binary vector pBI121 and pBin438 were cut by Xba I and BamH I to release the PAL fragment and the large fragments of pBI121 and pBin438. After ligation of the PAL fragment with pBI121 or pBin438 large fragment, we obtained two expressional vectors, named pBI121-PAL and pBin438-PAL respectively. The vectors were further transformed into Agrobacterium tumefeciens LBA4404.
The two constructions with sense and anti-sense direction of the PAL gene will be further introduced into poplar using Agrobacterium-mediated transformation to obtain the transgenic poplar plants. Both the PAL activity and the lignin content of the transgenic trees will be measured to study the effect on expression both sense and anti-sense PAL RNAs.
引文
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