中国科学院昆明动物研究所机构知识库

剪接体内含子（以下简称内含子）是普遍存在于真核生物基因内的非编码DNA序列，在基因发生转录后被剪接体去除而不出现在成熟的mRNA中。原核生物的基因组中不具有这类内含子，因此，内含子被认为是真核基因区别于原核基因的一个重要特征，也是原核生物进化成真核生物的过程中在基因组方面发生的一项重要变革。但由于从原核细胞进化成真核细胞的过程发生在遥远的远古，内含子的起源进化问题也就很难追溯以至一直未能解决。而发生在现存生物中的通过基因水平转移由原核生物进入到真核生物的原核基因，其内含子一般都会经历一个从无到有并随物种的分化而变化的过程。这或许给我们提供了一个探讨内含子起源进化的很好机会。绿藻中的果糖-1,6-二磷酸酶II （fructose-1,6-biphosphatase, FBPase II）基因就是这样一个基因。另外，已有越来越多的证据表明早期真核生物包括真核生物的最后共同祖先（Last Eukaryotic Common Ancestor, LECA）的基因组均含有丰富的内含子，因此，现存一些生物基因组中内含子极少的情况势必是因为发生了大量内含子丢失所致。但引起这样大规模内含子丢失的原因一直不得而知，而这些生物中的少量内含子在这种大规模的丢失过程中为何又能保留下来更是令人困惑。蓝氏贾第虫就是这样一个例子，其基因组中只有8个内含子，相对许多生物的成千上万的内含子来说是一个罕见情形。本论文就是分别利用绿藻中的FBPase II基因和贾第虫基因组为研究对象探讨内含子起源和进化的问题。首先，在我们的前期工作中发现，莱茵衣藻基因组中的FBPase II是一个从放线菌中通过基因水平转移得到的原核型基因。本文通过调查此基因在绿藻门许多代表物种中的分布情况并进行分子系统分析，发现FBPase II普遍存在于绿藻门这一类真核生物中，且它们在分子系统树上先聚为一支再与放线菌的FBPase II形成姐妹支。因此绿藻的FBPase II应该是在绿藻的共同祖先时期从放线菌水平转移而得到的。本研究收集鉴定了多个绿藻种类代表物种的FBPase II基因，从中鉴定出它们的内含子并进行系统的比较分析，发现：内含子的产生存在着相位和序列方面的偏好性——绝大多数的内含子都是产生在即使发生错误剪接和内含子滑动也不易产生不利影响的0相位，且明显地倾向于产生在插入于“G|G”这种proto-splice sites中。不同绿藻种类的基因组中的内含子密度差异较大，而不同绿藻中的FBPase II所含内含子的数量也是不同的，有趣的是我们发现某一绿藻的FBPase II基因中的内含子密度与该绿藻全基因组的内含子密度基本是一致的。这表明原核型基因在进入绿藻的共同祖先以后随着物种的分化，其内含子的变化并不具有统一模式，而是与其所在的绿藻物种基因组中内含子整体的变化相适调，最终达到密度上的一致性。进一步的分析发现其中引起变化的重要因素很可能是外显子的长度，这可能就是因为外显子的长度过长和过短都会影响到基因的正确剪接和加工。所以原核型的基因通过水平基因转移进入真核生物以后就要通过插入内含子的方式来缩短外显子的长度，以达到其所在的具体物种本身所能擅长处理的长度。以上结果提示，内含子在最初起源时，对相位和插入位点方面是存在偏好性的。而且进入到真核生物的原核基因中内含子产生的多寡与其所在的具体物种的整体基因组环境密切相关。其次，已知蓝氏贾第虫是一种基因组相对简单的寄生原虫，与许多真核生物成千上万的内含子不同其基因组中仅含八个内含子。所以贾第虫有可能作为研究内含子丢失的一个理想模型。本文首先通过对贾第虫中的内含子及内含子所在基因的保守性进行分析，发现贾第虫虽经历了内含子的大量丢失，但同时也存在新内含子的产生。随后我们对贾第虫内含子丢失的机制、原因行了分析研究，结果表明逆转录介导的内含子丢失应该是其发生内含子丢失的一条重要途；同时否定了贾第虫是因为寄生而造成内含子丢失的推测，但贾第虫内含子丢失的真正原因未能发现。对于这些少量的内含子为何在丢失的强大压力下能保留下来的问题，我们有了重要的发现：这些内含子的保留原来不是因为它们自身及其所在的基因所具有的任何特殊性质或重要功能所致，前人的“内含子功能限制其丢失”的推测至少在贾第虫中不成立；而有意思的是我们却发现贾第虫中部分含内含子的基因的互补链区域也是基因，二者形成顺-反式的基因重叠，并且内含子正好是位于该重叠区内。这表明这些内含子的保留应该正是其互补链的基因所限制的结果。这提示这些内含子之所以能被保留不一定是由于其本身的功能限制，而是受其本身以外的因素影响，来自其他基因或者基因组功能元件的“重叠限制”可能就是其中重要的一种原因。因此，本研究结果不仅可以为内含子和基因组的进化和功能研究提供一些新的线索，也提示我们在探索未知的基因组功能元件时，这种与内含子的重叠区域或许值得关注。

Spliceosomal intron (hereinafter referred to as an intron) are non-coding DNA sequences that ubiquitous in eukaryotic genes and cut out by the spliceosome after transcription and does not appear in mature mRNAs. Prokaryotes do not possess such introns in their genomes. Therefore, introns are considered to be an important feature distinguishing eukaryotic genes from prokaryotic genes, and an important change from prokaryotes to eukaryotes. However, since the process of evolution from prokaryotic cells to eukaryotic cells took place in the distant past, the origin and evolution of introns are difficult to trace back. The introns of prokaryotic genes that enter the eukaryotes from prokaryotic organisms through horizontal gene transfer in modern organism are generally experienced the process from origin to differentiation. Therefore, this may provide us with a good opportunity to explore the origin and evolution of introns. The fructose-1,6-bisphosphatase II (FBPase II) gene in Chlorophyta is such a gene. Besides, accumulating evidence suggests that the last eukaryotic common ancestor (LECA) is intron rich, and thus very few introns in some modern eukaryotes must be the consequence of massive loss. But the cause of such large-scale intron loss has been unknown, and it is more confusing why these small introns can survive in the process of large-scale loss. There are only eight introns in the entire genome of Giardia lamblia, this is a rare situation compared to the thousands of introns of many eukaryotes, which can be used as a good model to study these problems. In this work, we used the FBPase II gene in the green algae and the Giardia genome to study the problems about the origin and evolution of introns.Firstly, in our previous work, it was found that FBPase II in the genome of Chlamydomonas reinhardtii is a prokaryotic gene obtained by HGT from Actinomycetes. By investigating the distribution of this gene in many representativeall species of Chlorophyta and molecular phylogenetics, it was found that FBPase II is ubiquitous in Chlorophyta, and the FBPase II of Chlorophyta are clustered together then formed a sister clade with FBPase II of Actinomycetes in the phylogenetic tree, so the FBPase II in green alga is arise from Actinobacteria during the common ancestor of Chlorophyta. We collected and identified the FBPase II genes of several species of green algae, and then identified and conducted a systematic analysis about their introns. The analysis results show that prokaryotic genes show phase-biased and sequence-biased when inserting new introns --—the vast majority of introns are phase 0 that are less prone to adverse effects when mis-splicing and intron sliding occur; these introns tend to be inserted into the proto-splice sites of G|G. The density of intron in different algae genomes is different, and the number of introns gained in FBPase II in different algae is also different; interestingly, the intron density in each FBPase II is approximately the same as the intron density of the genome in which it is located. This indicates that the prokaryotic gene does not necessarily comply with some unified models when they obtain introns after enter the common ancestor of chlorophyta and differentiate with the species divergence, but according to the intron density in their respective recipient organisms. It is found that the influencing factors are likely to be the length of the exon in the genome of the eukaryotic receptor organism, so the exon cannot be too long or too short. Therefore, after the prokaryotic gene enters the eukaryote by HGT, the length of the exon will be shortened by inserting some introns to reach the length that that can be well handled by the eukaryotic receptor organisms. This may indicate that when introns are originally produced in eukaryotic genes, there is a preference for phase and insertion sites. Moreover, the amount of introns produced in prokaryotic genes entering eukaryotes is closely related to the overall genomic environment of the particular species in which they are located.Giardia is a parasitic protozoan with a relatively simple genome and few introns, Unlike thousands of introns in many eukaryotes, only eight spliceosomal introns were found in its genome. Therefore, Giardia can be used as an ideal model for studying the loss and retention of introns. Our investigation finds that despite of constant selective pressure of intron loss in Giardia’s evolution, intron gain still occurred through the conservative analysis of the intron and intron-containing genes in Giardia. Then a comprehensive analysis of the mechanism, causes of intron loss was carried out, it is shown that reverse transcription-mediated intron loss is an important way for Giardia intron loss, and it negates the hypothesis that parasitism caused the intron loss in Giardia, but the causes of the intron loss remain elusive despite extensive research. As for the few intron retention in Giardia, not finding any special features or functional importance of these introns and intron-containing genes responsible for their retention. Therefore, the “functional constraint” of introns themselves may at least not be applicable to the retention of the introns in Giardia. Interestingly, we found that the complementary region of some intron-containing genes are also genes, both the intron-containing gene and the gene in the complementary strand form sense-antisense gene pairs, and that the introns exactly reside in the overlapping regions. These observations suggest that the retention of these introns may be the result of the restriction of the genes of their complementary strands. This further suggests that the retention of introns is not necessarily due to functional constraint of the introns themselves but due to the causes outside of introns, and “overlap constraint” imposed by genes or other genomic functional elements may be an important one of the reasons. These findings may shed some new lights on intron evolution and/or function. Conversely, such an intron retention phenomenon probably can provide a valuable clue to find new genomic functional elements – in the overlapping area with introns.