技术领域technical field
本发明属于林业育种技术领域，具体为杂交杨树转基因育种的方法。The invention belongs to the technical field of forestry breeding, in particular to a method for transgenic breeding of hybrid poplars.
背景技术Background technique
杨树属于被子植物门(Angiosperms)双子叶植物纲(Dicots)杨柳科(Salicaceae)杨属(Populus)的落叶木本植物，杨树生长迅速，适应性广和柔韧性强，广泛应用于生态防护、城市绿化、工业和纤维生产。杨树除了具有分布广泛、生长迅速、产量高、环境适应能力强且易改良等诸多特点外，其全基因组测序已经完成，是林木遗传育种改良的理想模式植物。所有的杨树树种都具有无性繁殖的能力。杨树是根癌农杆菌的天然寄主，便于利用根癌农杆菌介导法进行植物的遗传转化，因此杨树被认为是林木基因工程研究的模式植物之一，此外杨树及其杂种被认为是生物能源原料生产的首选多年生植物。杨树是温带地区生长速度最快的树种，具有多种农艺性状和物种优势，目前的生物量增长率接近10-15kg/hm2每年；生物量可以储存在根部通过灌溉萌芽再生，可以最大限度地减少对储藏设施的投入；杨树与其他纤维作物相比耕作和收割的周期更短，对温室气体排放的净效应非常有利。Poplar belongs to the deciduous woody plants of Angiosperms, Dicots, Salicaceae and Populus. Poplar grows rapidly, has wide adaptability and flexibility, and is widely used in ecological protection. , urban greening, industry and fiber production. In addition to the characteristics of wide distribution, rapid growth, high yield, strong environmental adaptability and easy improvement, poplar is an ideal model plant for forest genetic breeding and improvement. All poplar species are capable of asexual reproduction. Poplar is the natural host of Agrobacterium tumefaciens, and it is convenient to use the Agrobacterium tumefaciens-mediated method for genetic transformation of plants. Therefore, poplar is considered to be one of the model plants for forest genetic engineering research. In addition, poplar and its hybrids are considered It is the perennial plant of choice for bioenergy feedstock production. Poplar is the tree species with the fastest growth rate in temperate regions. It has a variety of agronomic traits and species advantages. The current biomass growth rate is close to 10-15kg/hm2 per year; The investment in storage facilities is greatly reduced; poplar has a shorter cycle of tilling and harvesting than other fiber crops, and the net effect on greenhouse gas emissions is very beneficial.
‘717’杂交杨(Populus tremula×P.alba‘INRA 717-1B4’)是从欧洲山杨和银白杨的杂交群体中选育出的优良品种，具有较强的耐逆性，对植被恢复、防止水土流失和盐碱地修复具有很大的应用价值，还可用于生物质能源和纤维能源开发。‘717’杂交杨是多年生植物，相比于水稻(Oryza sativa)和拟南芥(Arabidopsis thaliana)等一年生草本植物在研究木材的形成和季节性休眠等方面具有独特的优势，而且杨树在进化过程中产生大量重复的序列其调控机制更为复杂，因此以‘717’杂交杨为研究对象进行基因功能分析显得尤为重要。'717' hybrid poplar (Populus tremula×P.alba'INRA 717-1B4') is an excellent variety bred from the hybrid population of Populus tremula and Populus alba. Loss and saline-alkali restoration have great application value, and can also be used in the development of biomass energy and fiber energy. The '717' hybrid poplar is a perennial plant. Compared with annual herbaceous plants such as rice (Oryza sativa) and Arabidopsis thaliana (Arabidopsis thaliana), it has unique advantages in the study of wood formation and seasonal dormancy. The regulatory mechanism of a large number of repeated sequences generated in the process is more complex, so it is particularly important to analyze the gene function of the '717' hybrid poplar as the research object.
发明内容Contents of the invention
本发明公开了84K杨树的ARK1基因，其特征在于ARK1基因的序列如核苷酸序列如SEQ.ID.NO.1所示。The invention discloses the ARK1 gene of 84K poplar, which is characterized in that the sequence of the ARK1 gene is as shown in SEQ.ID.NO.1 as the nucleotide sequence.
84K杨树的ARK1基因的运用，培育转ARK1基因的‘717’杂交杨。The use of the ARK1 gene of 84K poplar to breed the '717' hybrid poplar with the ARK1 gene.
84K杨树的ARK1基因的运用在于构建了ARK1基因(来自84K杨树)的转基因载体并通过转基因获得了转ARK1基因的‘717’杨树植株。本发明的转基因杂交杨在节间距、茎粗、叶柄长、叶宽、叶长和苗高上有显著差异，在植物的生长发育和木质素调控的相关基因上有差异。84K杨树的ARK1基因参与了植物的生长过程，是一个重要的与生长相关的基因，在作物种质资源改良、遗传育种方面具有广阔的应用前景。The application of the ARK1 gene of 84K poplar is to construct the transgenic vector of ARK1 gene (from 84K poplar) and obtain the '717' poplar plant of transgenic ARK1 gene through transgenesis. The transgenic hybrid poplar of the present invention has significant differences in internode distance, stem diameter, petiole length, leaf width, leaf length and seedling height, and has differences in plant growth and development and related genes regulated by lignin. The ARK1 gene of 84K poplar is involved in the growth process of plants. It is an important growth-related gene and has broad application prospects in crop germplasm resource improvement and genetic breeding.
在本发明中，84K杨树的ARK1基因，构建了新的载体，并培育了转ARK1基因的‘717’杂交杨，生长素相关基因上调表达，会促进植株生长；同时木质素合成相关基因下调表达，会导致木质素含量下降，提高其工业利用价值。In the present invention, the ARK1 gene of 84K poplar is used to construct a new carrier, and the '717' hybrid poplar with ARK1 gene is bred, and the expression of auxin-related genes is up-regulated, which will promote plant growth; at the same time, lignin synthesis-related genes are down-regulated Expression will lead to a decrease in lignin content and increase its industrial utilization value.
附图说明Description of drawings
图1是PCR检测图，图中M为D2000 maker；CK是非转基因‘717’杂交杨植株的DNA为模板的PCR产物；1-11分别是以转基因‘717’杂交杨不同植株的DNA为模板的PCR产物；Figure 1 is a PCR detection map, in which M is D2000 maker; CK is the PCR product of the DNA of the non-transgenic '717' hybrid poplar plant as a template; 1-11 are respectively based on the DNA of different transgenic '717' hybrid poplar plants as templates PCR product;
图2是非转基因‘717’杂交杨和转ARK1基因‘717’杂交杨形态图，A：非转基因‘717’杂交杨；B与C：表示转ARK1基因‘717’杂交杨；Figure 2 is the morphological diagram of non-transgenic '717' hybrid poplar and transgenic ARK1 gene '717' hybrid poplar, A: non-transgenic '717' hybrid poplar; B and C: transgenic ARK1 gene '717' hybrid poplar;
图3显微观察细胞图A,B为非转基因‘717’杂交杨；C,D转ARK1基因‘717’杂交杨；Figure 3 Microscopic observation of cells A, B are non-transgenic ‘717’ hybrid poplar; C, D transgenic ARK1 gene ‘717’ hybrid poplar;
图4显示基因表达差异。Figure 4 shows gene expression differences.
具体实施方式Detailed ways
步骤1、引物设计和合成Step 1. Primer design and synthesis
1)目的基因扩增引物1) Target gene amplification primers
引物ARBF，序列5′-GGAGAGGACACGCTCGAGATGGAGGGTGGTGATGGTG-3′，长度37bp，序列如SEQ.ID.NO.2所示。Primer ARBF, sequence 5'-GGAGAGGACACGCTCGAGATGGAGGGTGGTGATGGTG-3', length 37bp, sequence shown in SEQ.ID.NO.2.
引物ARBR，序列5′-ATCCTTGTAGTCGAATTCAAGCAGTGTGGGAGAGATGTC-3′，长度39bp，序列如SEQ.ID.NO.3所示。Primer ARBR, sequence 5'-ATCCTTGTAGTCGAATTCAAGCAGTGTGGGAGAGATGTC-3', 39bp in length, the sequence is shown in SEQ.ID.NO.3.
2)目的基因扩增及胶回收，使用百泰克胶回收试剂盒回收目的基因条带。2) Amplify the target gene and recover the gel, and recover the band of the target gene using the Biotech gel recovery kit.
3)目的基因与PART-CAM-FLAG连接，构建载体。3) The target gene is connected with PART-CAM-FLAG to construct a vector.
PART-CAM-FLAG-gene构建方案：PART-CAM-FLAG-gene construction scheme:
1)用XhoI和EcoRI双酶切质粒PART-CAM-FLAG，回收大片段；1) Digest the plasmid PART-CAM-FLAG with XhoI and EcoRI to recover the large fragment;
双酶切反应体系：Double enzyme digestion reaction system:
37℃酶切2.5h；65℃：20min失活。Digestion at 37°C for 2.5h; 65°C: 20min for inactivation.
2)用上游和下游引物克隆目的基因，用XhoI和EcoRI双酶切PCR产物，回收小片段；2) Cloning the target gene with upstream and downstream primers, double-digesting the PCR product with XhoI and EcoRI, and recovering small fragments;
3)将1、2所得片段用重组连接酶重组连接、转化至DH5α感受态细胞；菌落PCR检测；3) The fragments obtained in 1 and 2 were recombined and connected with a recombinant ligase, and transformed into DH5α competent cells; colony PCR detection;
4)挑取阳性菌落测序验证，获得构建成功的载体及相关菌株。4) Pick positive colonies for sequencing verification, and obtain successfully constructed vectors and related strains.
因部分载体会与外源基因ARK1基因连在一起，部分不会与外源基因连在一起，部分还处于线性状态，部分会处于环状状态，只有含外源基因并处于环状状态的载体才是有效并可在大肠杆菌中扩增的载体，因而需要挑选阳性克隆。构建的载体在大肠杆菌中比较容易扩增，而且容易筛选到含外源基因ARK1基因并呈环状的载体。但用大杨杆菌无法侵染杨树，只有根癌农杆菌才能侵染杨树。所以要筛选到含ARK1基因载体的大肠杆菌，并扩增到足够的量，并分离出来转化到根癌农杆菌，才能进行下一步的侵染。Because some vectors will be connected with the exogenous gene ARK1 gene, some will not be connected with the foreign gene, some will still be in a linear state, and some will be in a circular state, only the carrier containing the foreign gene and in a circular state It is an effective vector that can be amplified in Escherichia coli, so it is necessary to select positive clones. The constructed vector is relatively easy to amplify in Escherichia coli, and it is easy to screen out the circular vector containing the foreign gene ARK1 gene. However, poplar trees cannot be infected with E. poplar, and only Agrobacterium tumefaciens can infect poplar trees. Therefore, the Escherichia coli containing the ARK1 gene carrier must be screened, amplified to a sufficient amount, and isolated and transformed into Agrobacterium tumefaciens before the next step of infection can be carried out.
步骤2、载体转化根癌农杆菌Step 2, vector transformation of Agrobacterium tumefaciens
从含ARK1基因载体的DH5α中提质粒，并转化根癌农杆菌菌株LBA4404，PCR扩增挑选阳性菌株，用于下一步骤中浸染。The plasmid was extracted from DH5α containing the ARK1 gene vector, and transformed into Agrobacterium tumefaciens strain LBA4404, and positive strains were selected by PCR amplification for the next step of infection.
步骤3、杂交杨转基因及检测Step 3, transgenic and detection of hybrid poplar
1)‘717’杂交杨组培体系的建立1) Establishment of '717' hybrid poplar tissue culture system
用75％酒精处理15s，0.1％升汞处理10min进行外植体消毒；在诱导愈伤组织时，所用培养基为MS+1.0mg/L6-BA+1.2mg/l NAA。不定芽分化配方为MS+1.0mg/l6-BA+0.4mg/lZT。幼苗生根的配方是1/2MS+0.02mg/L NAA+0.6mg/LIBA。Treat the explants with 75% alcohol for 15 seconds and 0.1% mercury chloride for 10 minutes to disinfect the explants; when inducing callus, the medium used is MS+1.0mg/L6-BA+1.2mg/l NAA. The formula for adventitious bud differentiation is MS+1.0mg/l6-BA+0.4mg/lZT. The formula for seedling rooting is 1/2MS+0.02mg/L NAA+0.6mg/LIBA.
2)构建‘717’杂交杨叶片遗传转化体系2) Construct the genetic transformation system of '717' hybrid poplar leaves
在建立‘717’杂交杨叶片组培体系的基础上,确定‘717’杂交杨叶片Kan浓度的选择压为100mg/L，羧苄青霉素最佳抑菌浓度为200mg/L。菌液浓度OD600值为0.3，浸染时间为10min时，农杆菌的转化杂交杨叶片效果最好，转化方法为叶盘法。包括如下步骤：On the basis of establishing the tissue culture system of '717' hybrid poplar leaves, the selection pressure of Kan concentration in '717' hybrid poplar leaves was determined to be 100 mg/L, and the optimal inhibitory concentration of carbenicillin was 200 mg/L. When the OD600 value of the bacterial solution concentration was 0.3 and the dipping time was 10 minutes, the Agrobacterium transformed the hybrid poplar leaves with the best effect, and the transformation method was the leaf disc method. Including the following steps:
a)侵染a) Infestation
于超净工作台上，从培养瓶中取出经预培养的‘717’杂交杨叶片，放入前期经过活化的菌液中浸泡一段时间。然后取出‘717’杂交杨外植体置于无菌纸上吸去附着的菌液。On the ultra-clean workbench, take out the pre-cultivated '717' hybrid poplar leaves from the culture bottle, and soak them in the pre-activated bacterial solution for a period of time. Then the '717' hybrid poplar explants were taken out and placed on sterile paper to absorb the attached bacterial solution.
b)共培养b) Co-cultivation
将用农杆菌侵染过的‘717’杂交杨叶片接种在愈伤组织诱导培养基(MS+NAA1.0mg/L+6-BA 1.0mg/L)上，在28℃暗培养条件下共培养2～4d，可见到叶片周围生成农杆菌菌斑。The '717' hybrid poplar leaves infected with Agrobacterium were inoculated on the callus induction medium (MS+NAA 1.0mg/L+6-BA 1.0mg/L) and co-cultivated at 28°C in the dark From 2 to 4 days, Agrobacterium plaques can be seen around the leaves.
c)选择培养c) Selective culture
将经过共培养的愈伤组织用无菌水清洗3次左右，用无菌纸吸干水分，然后转移到加有选择压(羧苄青霉素+Kan)的脱菌分化培养基(MS+1.0mg/L 6-BA+0.4mg/L ZT)上，在光照周期16/8h、28℃条件下进行选择培养。Wash the co-cultured callus about 3 times with sterile water, dry the water with sterile paper, and then transfer it to the sterile differentiation medium (MS+1.0mg /L 6-BA+0.4mg/L ZT), the selection culture was carried out under the conditions of light cycle 16/8h and 28°C.
d)继代选择培养d) Subculture selection culture
28d左右，更换一次选择培养基诱导其分化，诱导新的愈伤组织和出芽。About 28 days, change the selection medium once to induce its differentiation, induce new callus and sprout.
e)生根培养e) Rooting culture
待不定芽长到2～3cm时，移到含有选择压(羧苄青霉素+Kan)的生根培养基(1/2MS+0.02mg/L NAA+0.6mgL IBA)中进行生根培养，待植物长出不定根后，随后移至温室练苗。When the adventitious buds grow to 2-3cm, move them to the rooting medium (1/2MS+0.02mg/L NAA+0.6mgL IBA) containing selective pressure (carbenicillin+Kan) for rooting culture, and wait for the plants to grow After adventitious rooting, it was then moved to the greenhouse to practice seedlings.
3)转基因植株的分子检测3) Molecular detection of transgenic plants
根据构建的表达载体，设计相应的特异检测引物，上游引物则是ARK1目基因内部的序列，下游引物采用了载体NPTⅡ中的序列，引物的序列为：According to the constructed expression vector, design corresponding specific detection primers, the upstream primer is the sequence inside the ARK1 gene, and the downstream primer uses the sequence in the vector NPTII, the sequence of the primer is:
Forward 5’-AAGATCCAGCCCTTGACCAA-3′序列如SEQ.ID.NO.4所示；Forward 5'-AAGATCCAGCCCTTGACCAA-3' sequence is shown in SEQ.ID.NO.4;
Reverse 5’-CATTGCCATCACCACAACCA-3′序列如SEQ.ID.NO.5所示The sequence of Reverse 5'-CATTGCCATCACCACAACCA-3' is shown in SEQ.ID.NO.5
利用上述引物，按照下列体系进行PCR反应。Using the above primers, a PCR reaction was carried out according to the following system.
分别加入上述各成分后，进行短暂离心将液体收集到管底，使用Takara公司的Prime STAR Max Premix DNA聚合酶来进行PCR反应。After adding the above-mentioned components respectively, perform short centrifugation to collect the liquid to the bottom of the tube, and use Takara's Prime STAR Max Premix DNA polymerase to perform PCR reaction.
PCR扩增程序如下：The PCR amplification procedure is as follows:
98℃，3min；35Cycles；94℃30s，55℃，30s；72℃5min；72℃10min。98°C, 3min; 35Cycles; 94°C, 30s, 55°C, 30s; 72°C, 5min; 72°C, 10min.
然后进行PCR反应，PCR反应步骤与菌落PCR一致。然后用1.2％琼脂糖凝胶(0.5μg/mol溴化乙锭)电泳对PCR扩增产物进行检测。Then carry out PCR reaction, and the steps of PCR reaction are consistent with colony PCR. Then use 1.2% agarose gel (0.5μg/mol ethidium bromide) electrophoresis to detect the PCR amplification products.
提取转ARK1基因的‘717’杂交杨叶片的DNA，进行PCR检测，发现阳性率为31.43％，初步证明ARK1过表达载体遗传转化‘717’杂交杨成功(如图1所示)。The DNA of the leaves of the ‘717’ hybrid poplar transformed with the ARK1 gene was extracted and tested by PCR, and the positive rate was found to be 31.43%, preliminarily proving that the genetic transformation of the ‘717’ hybrid poplar with the ARK1 overexpression vector was successful (as shown in Figure 1).
4)转基因植株形态学变化检测4) Detection of morphological changes in transgenic plants
对‘717’杂交杨转基因植株与非转基因植株的组培苗的形态学差异做比较，测量相同生长周期的茎段粗细、节间数和节间长度，每组做3次重复。The morphological differences between the transgenic plants of '717' hybrid poplar and non-transgenic plants were compared, and the thickness of stem segments, the number of internodes and the length of internodes in the same growth cycle were measured, and each group was repeated three times.
结果表明：转基因‘717’杂交杨苗相比于非转基因苗在节间距、茎粗、叶柄长、叶宽、叶长和苗高上有显著差异。转基因‘717’杂交杨苗茎段细长，通常呈束状与多分枝，没有细长的叶柄，叶子形状细长，有的叶片未发育完全(如图2所示)。The results showed that the internode distance, stem diameter, petiole length, leaf width, leaf length and seedling height of transgenic '717' hybrid poplar seedlings were significantly different from those of non-transgenic seedlings. The stems of transgenic '717' hybrid poplar seedlings are slender, usually fascicle-shaped and multi-branched, without slender petioles, and the leaves are slender, and some leaves are not fully developed (as shown in Figure 2).
5)石蜡切片及显微结构观察5) Paraffin section and microstructure observation
a)取材：迅速切取‘717’杂交杨第5片叶之下的茎段，长约3～5mm，立即浸入FAA固定液(50.0％的酒精，5.0％乙酸，3.7％的甲醛，41.3％的水)。a) Material collection: quickly cut the stem section under the 5th leaf of '717' hybrid poplar, about 3-5mm long, and immediately immerse in FAA fixative solution (50.0% alcohol, 5.0% acetic acid, 3.7% formaldehyde, 41.3% water).
b)固定：在FAA固定液中室温下固定24h以上。b) Fixation: fix in FAA fixative solution at room temperature for more than 24 hours.
c)脱水：先用蒸馏水洗涤；随后用叔丁醇与酒精的混合液进行梯度脱水，顺序为70％-85％-95％-100％-100％，每步2h。抽气45min×8，逐级脱水至100％叔丁醇，100％叔丁醇脱水3h，重复3次。c) Dehydration: wash with distilled water first; then carry out gradient dehydration with a mixture of tert-butanol and alcohol, the order is 70%-85%-95%-100%-100%, each step is 2h. Evacuate for 45min×8, dehydrate step by step to 100% tert-butanol, dehydrate 100% tert-butanol for 3h, repeat 3 times.
d)浸蜡：在‘717’杂交杨样品中加入融化的石蜡，在65℃的温箱中放置24h以上。d) Wax immersion: Add melted paraffin to the '717' hybrid poplar sample, and place it in an incubator at 65°C for more than 24 hours.
e)包埋：将65℃融化的石蜡倒入3cm×3cm的纸盒内，等石蜡稍微凝固，底部见白时，将‘717’杂交杨材料竖插到石蜡里，用解剖针将气泡挑除，静放待蜡块凝固后，放5℃冰箱保存待用。e) Embedding: Pour 65°C melted paraffin into a 3cm×3cm carton, wait for the paraffin to solidify slightly, and when the bottom is white, insert the '717' hybrid poplar material vertically into the paraffin, and pick out the air bubbles with a dissecting needle Remove, let it stand until the wax block is solidified, and store it in a refrigerator at 5°C for later use.
f)切片：修蜡块，粘于木块上，用切片机切8μm厚的切片，将切片用沾片剂粘在载玻片上，载玻片在45℃度温箱中过夜，也可以室温晾干载玻片。f) Slicing: wax repair block, glued to the wooden block, cut 8 μm thick slices with a microtome, stick the slices on the glass slide with a dipping agent, and put the slide in a temperature box at 45°C overnight, or at room temperature Allow slides to dry.
g)脱蜡：切片干后经二甲苯→二甲苯→1/2二甲苯+1/2酒精→2次100％酒精→95％酒精→95％酒精→70％酒精→50％酒精→蒸馏水1min。g) Dewaxing: After the slices are dried, pass through xylene → xylene → 1/2 xylene + 1/2 alcohol → 2 times 100% alcohol → 95% alcohol → 95% alcohol → 70% alcohol → 50% alcohol → distilled water for 1 min .
h)染色：苏木精染色10min，50％酒精→70％酒精→85％酒精→95％酒精→70％酒精→2次100％酒精→1/2二甲苯+50％酒精→2次二甲苯h) Staining: Hematoxylin staining for 10 minutes, 50% alcohol→70% alcohol→85% alcohol→95% alcohol→70% alcohol→2 times 100% alcohol→1/2 xylene+50% alcohol→2 times xylene
i)镜检：封片，显微镜观察切片。i) Microscopic examination: cover the slices, and observe the slices under a microscope.
结果表明：石蜡切片及显微结构观察转基因‘717’杂交杨苗较非转基因苗茎部木质部与韧皮部细胞数量减少和细胞直径变小(如图3所示)。The results showed that the number of xylem and phloem cells in the stem of the transgenic '717' hybrid poplar seedlings were reduced and the cell diameters were smaller than those of non-transgenic seedlings (as shown in Figure 3) by paraffin section and microstructural observation.
4、转录组测序4. Transcriptome sequencing
提取转基因‘717’杂交杨与非转基因‘717’杂交杨样品的mRNA，反转录成cDNA，利用测序平台HiSeq对转基因‘717’杂交杨与非转基因‘717’杂交杨样品的cDNA进行高通量测序分析，包括测序数据过滤、差异表达基因GO功能分析和Pathway功能分析，进行相关差异表达基因的筛选与分析。The mRNA of transgenic '717' hybrid poplar and non-transgenic '717' hybrid poplar samples was extracted, reverse-transcribed into cDNA, and the cDNA of transgenic '717' hybrid poplar and non-transgenic '717' hybrid poplar samples were analyzed by high-pass sequencing using the sequencing platform HiSeq Quantitative sequencing analysis, including sequencing data filtering, GO function analysis of differentially expressed genes and Pathway function analysis, to screen and analyze related differentially expressed genes.
结果表明：原始数据经过质控过滤，去除冗余序列和低质量值的reads后，共获得到45.8GB的Clean reads，其中测序质量Q30碱基百分比在91.85％及以上，四个样品中平均GC含量为47.69％，说明测序质量良好，满足建库要求。同时将所测序得到的Clean reads与杨树参考基因组进行序列比对后，比对效率从55.61％到60.61％不等，约有57.53％的序列可被参考基因组注释。其中，与参考序列唯一比对位置的Clean reads比对率为56.61％。在差异表达基因集基因数目中，筛选出641条差异表达基因，其中上调基因数目有389个，下调基因有252个(如图4所示)。The results show that: after the original data is filtered by quality control, redundant sequences and low-quality reads are removed, a total of 45.8GB of Clean reads are obtained, of which the sequencing quality Q30 base percentage is 91.85% and above, and the average GC among the four samples The content was 47.69%, indicating that the sequencing quality was good and met the requirements for library construction. At the same time, after comparing the sequenced Clean reads with the poplar reference genome, the comparison efficiency ranged from 55.61% to 60.61%, and about 57.53% of the sequences could be annotated by the reference genome. Among them, the Clean reads alignment rate of the unique alignment position with the reference sequence was 56.61%. In the number of genes in the differentially expressed gene set, 641 differentially expressed genes were screened out, including 389 up-regulated genes and 252 down-regulated genes (as shown in Figure 4).
通过对与细胞尖端生长和分生组织生长的调节与通路植物激素信号转导和苯丙烷类生物合成通路中显著差异表达的基因进行分析，经差异基因功能注释后，我们筛选得到27个参与杨树生长过程中的差异表达基因，包括蛋白类、转录因子类和蛋白激酶类，这些基因与植物的生长发育和木质素调控相关。在转基因植株中，植物生长相关的基因表达大多呈上调状态，这样意味着在转ARK1‘717’杂交杨中与生长相关基因上调，说明ARK1基因对植物的生长有正向调控作用；而苯丙烷途径中木质素合成相关酶如羟苯基木质素(P-Hydroxyphenyl Lignin)、愈创木基木质素(Guaiacyl lignin)、5-羟基愈创木基木质素(5-Hydroxyguaiacyl lignin)、紫丁香基木质素(Syringyl lignin)、对香豆酸(p-Coumaricacid)和对香豆酰基辅酶A(p-Coumaroyl-CoA)等的表达均下调。而且植株的表现型出现高度、直径等方面的异常生长。By analyzing the significantly differentially expressed genes related to the regulation and pathway of cell tip growth and meristem growth, phytohormone signal transduction and phenylpropanoid biosynthesis pathway, after functional annotation of differential genes, we screened and obtained 27 genes involved in poplar Differentially expressed genes during tree growth, including proteins, transcription factors and protein kinases, which are related to plant growth and development and lignin regulation. In the transgenic plants, the expression of genes related to plant growth was mostly up-regulated, which means that the genes related to growth in the transgenic ARK1'717' hybrid poplar were up-regulated, indicating that the ARK1 gene has a positive regulatory effect on plant growth; while phenylpropane Enzymes related to lignin synthesis in the pathway, such as P-Hydroxyphenyl Lignin, Guaiacyl lignin, 5-Hydroxyguaiacyl lignin, Syringyl The expressions of lignin (Syringyl lignin), p-coumaric acid (p-Coumaric acid) and p-coumaroyl-CoA (p-Coumaroyl-CoA) were down-regulated. Moreover, the phenotype of the plants showed abnormal growth in height and diameter.
木质素是木材的三大主要化学成分之一(木质素、纤维素和半纤维素)，在植物中具有重要的生物学功能。木质素限制了造纸工业的发展，这是由于造纸过程中会产生环境污染和需要大量的木材生产能源造成的，树木中木质素含量的降低不仅可以提高纸浆和造纸工业的经济和环境效益，还可以促进木质纤维素的分解，提高糖的转化效率。Lignin is one of the three major chemical constituents of wood (lignin, cellulose, and hemicellulose), which has important biological functions in plants. Lignin limits the development of the paper industry, which is caused by environmental pollution and the need for a large amount of wood production energy in the papermaking process. The reduction of lignin content in trees can not only improve the economic and environmental benefits of the pulp and paper industry, but also It can promote the decomposition of lignocellulose and improve the conversion efficiency of sugar.
综上，本转基因研究可提高杨树的生长率及其在造纸工业领域的价值。In summary, this transgenic research can improve the growth rate of poplar and its value in the field of paper industry.
<110> 西南林业大学<110> Southwest Forestry University
<120> 84K杨树ARK1基因及其在杂交杨中的运用<120> 84K poplar ARK1 gene and its application in hybrid poplar
<160> 5<160> 5
<210> 1<210> 1
<211> 1352bp<211> 1352bp
<212> DNA<212>DNA
<213> 84K杨树<213> 84K Poplar
<400> 1<400> 1
TATCATGCGATCATAGGCGTCTCGCATATCTCATTAAAGCAGGACTCTAGATTAGGGACGTCTCTTATCGTCATCGTCCTTATAGTCTTGCTTGTCATCGTCATCTTTATAATCGCCCTTGTCATCGTCATCCTTGTAGTCGAATTCAAGCAGTGTGGGAGAGATGTCCATTGGGAAAGGATTGCCCAAAACATTATCCATGTAGTAATGAGGATGGCCGGCATCCATCACCACAAACTGCATGTCTTCCGATGGTTTCCAGTGTCGTTTCCTTTGGTTAATGAACCAGTTGTTTATTTGCTTCTGATCCAGACCAGTGGATTCAGCAAGGGCCAGCTTCTGCGACTCCGATGGATATGGCCATTTGTAATGCCTGCTCCACCAATCTAGCAGCTGTTGCCTGGCTTCTTTCGGTAACTTCCCTTTCTTTCTCTTTTTCATGAATTCCTGCTTGAGACTCCCTAAATATCCACTGTACCTGCGAAGTAGCTGACCCTTCAGTTCTTGATCTTCAGCTTGAGGATCTATGAAGTTGTTATTCACATCAACCTCTTCTTCAGATGACACATTCCTATCATTGCCATCACCACAACCAGAAATTGGAGAGGAAACAGTGAGAGCTTTGAATTGACACTCAACCCTCTGAAGAAAAAGCATGGCTTCCTTTAAGGGTTTAGAGAGTTCTTGCTCATACTTGGTCAGCATCTCACAGTAAGCCTCCATGAATTGGTCAAGGGCTGGATCTTCGCCGATGCAGCCTGTGTTAGCGGGGGCCATAGAACCAGCCGATGCGCAAGCTTCTTCTAGCCTAGCCACCACTTCAGGTGGTGCTCCAACCTTCTGACAATTAGCATAGGCAGCAAGGAGACGATGGTAGTGAGGATGAGCCATAATTTTGGCCTTCACAGAAGAACTACTACCATCATTGTTATCCATGAAATAACACCCAGTAGCAGTGACAGTGTTGTTGTGGTTGTGATCGTCAATAATCATAGAAGAAGAGCCACTAGCATTACTATGGATACGGTTATGGCCTTGATTGTTTGTAGGAGGTAGAGGAAGGAATAAGGTATTTGATATTGACGAATCGCCCTCATTAGCCCGATGATGATGATGATGATGATGAGCAGAAGACGAAGAAGACATGAGAGGCGTCATCATCATCATAGGGCAAAGTCCATTGCTGTTGTCTCCAAAAGCCATCATACATGAAGTGGTACTTGAACCACCATCACCACCCTCCATCTCGAGCGTGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATGTCACATATCATGCGATCATAGGCGTCTCGCATATCTCATTAAAGCAGGACTCTAGATTAGGGACGTCTCTTATCGTCATCGTCCTTATAGTCTTGCTTGTCATCGTCATCTTTATAATCGCCCTTGTCATCGTCATCCTTGTAGTCGAATTCAAGCAGTGTGGGAGAGATGTCCATTGGGAAAGGATTGCCCAAAACATTATCCATGTAGTAATGAGGATGGCCGGCATCCATCACCACAAACTGCATGTCTTCCGATGGTTTCCAGTGTCGTTTCCTTTGGTTAATGAACCAGTTGTTTATTTGCTTCTGATCCAGACCAGTGGATTCAGCAAGGGCCAGCTTCTGCGACTCCGATGGATATGGCCATTTGTAATGCCTGCTCCACCAATCTAGCAGCTGTTGCCTGGCTTCTTTCGGTAACTTCCCTTTCTTTCTCTTTTTCATGAATTCCTGCTTGAGACTCCCTAAATATCCACTGTACCTGCGAAGTAGCTGACCCTTCAGTTCTTGATCTTCAGCTTGAGGATCTATGAAGTTGTTATTCACATCAACCTCTTCTTCAGATGACACATTCCTATCATTGCCATCACCACAACCAGAAATTGGAGAGGAAACAGTGAGAGCTTTGAATTGACACTCAACCCTCTGAAGAAAAAGCATGGCTTCCTTTAAGGGTTTAGAGAGTTCTTGCTCATACTTGGTCAGCATCTCACAGTAAGCCTCCATGAATTGGTCAAGGGCTGGATCTTCGCCGATGCAGCCTGTGTTAGCGGGGGCCATAGAACCAGCCGATGCGCAAGCTTCTTCTAGCCTAGCCACCACTTCAGGTGGTGCTCCAACCTTCTGACAATTAGCATAGGCAGCAAGGAGACGATGGTAGTGAGGATGAGCCATAATTTTGGCCTTCACAGAAGAACTACTACCATCATTGTTATCCATGAAATAACACCCAGTAGCAGTGACAGTGTTGTTGTGGTTGTGATCGTCAATAATCATAGAAGAA GAGCCACTAGCATTACTATGGATACGGTTATGGCCTTGATTGTTTGTAGGAGGTAGAGGAAGGAATAAGGTATTTGATATTGACGAATCGCCCTCATTAGCCCGATGATGATGATGATGATGATGAGCAGAAGACGAAGAAGACATGAGAGGCGTCATCATCATCATAGGGCAAAGTCCATTGCTGTTGTCTCCAAAAGCCATCATACATGAAGTGGTACTTGAACCACCATCACCACCCTCCATCTCGAGCGTGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATGTCACA
<110> 西南林业大学<110> Southwest Forestry University
<120> 84K杨树ARK1基因及其在杂交杨中的运用<120> 84K poplar ARK1 gene and its application in hybrid poplar
<160> 5<160> 5
<210> 2<210> 2
<211> 37bp<211> 37bp
<212> DNA<212>DNA
<213> 84K杨树<213> 84K Poplar
<400> 2<400> 2
5′GGAGAGGACACGCTCGAGATGGAGGGTGGTGATGGTG-3′5'GGAGAGGACACGCTCGAGATGGAGGGTGGTGATGGTG-3'
<110> 西南林业大学<110> Southwest Forestry University
<120> 84K杨树ARK1基因及其在杂交杨中的运用<120> 84K poplar ARK1 gene and its application in hybrid poplar
<160> 5<160> 5
<210> 3<210> 3
<211> 39bp<211> 39bp
<212> DNA<212>DNA
<213> 84K杨树<213> 84K Poplar
<400> 3<400> 3
5′ATCCTTGTAGTCGAATTCAAGCAGTGTGGGAGAGATGTC -3′5′ATCCTTGTAGTCGAATTCAAGCAGTGTGGGAGAGATGTC-3′
<110> 西南林业大学<110> Southwest Forestry University
<120> 84K杨树ARK1基因及其在杂交杨中的运用<120> 84K poplar ARK1 gene and its application in hybrid poplar
<160> 5<160> 5
<210> 4<210> 4
<211> 20bp<211> 20bp
<212> DNA<212>DNA
<213> 84K杨树<213> 84K Poplar
<400> 4<400> 4
5’ -AAGATCCAGCCCTTGACCAA-3′5'-AAGATCCAGCCCTTGACCAA-3'
<110> 西南林业大学<110> Southwest Forestry University
<120> 84K杨树ARK1基因及其在杂交杨中的运用<120> 84K poplar ARK1 gene and its application in hybrid poplar
<160> 5<160> 5
<210> 5<210> 5
<211> 20bp<211> 20bp
<212> DNA<212>DNA
<213> 载体PART-CAM的NPTⅡ基因<213> NPTⅡ gene of vector PART-CAM
<400> 5<400> 5
5’ -CATTGCCATCACCACAACCA-3′5'-CATTGCCATCACCACAACCA-3'
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| CN201910855290.0ACN110527688A (en) | 2019-09-10 | 2019-09-10 | 84K poplar ARK1 gene and its utilization in Hybrid Poplar |
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| CB03 | Change of inventor or designer information | Inventor after:Zhang Hanyao Inventor after:Ye Qinxia Inventor after:Liu Xiaozhen Inventor after:Wei Zhuo Inventor after:Zhang Zhiming Inventor after:Bian Wen Inventor before:Zhang Hanyao Inventor before:Li Yipei Inventor before:Liu Xiaozhen Inventor before:Wei Zhuo Inventor before:Zhang Zhiming Inventor before:Bian Wen | |
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