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Eye and Vision 2020年第1期7卷 266-274页

作者： Tanner J.Ferguson Zachary Dockter Adam Bleeker Kayla L.Karpuk Justin Schweitzer Mitch J.Ibach John P.Berdahl Cole Eye Institute Cleveland ClinicClevelandOHUSA University of South Dakota Sanford School of Medicine Sioux FallsSDUSA Cleveland Eye Clinic ClevelandOHUSA Vance Thompson Vision Sioux FallsSDUSA

Background:Retrospective,consecutive case series to evaluate the implantation of two second-generation trabecular microbypass stents in combination with cataract surgery in a real-world,clinical setting.Methods:The series included 56 eyes implanted with the iStent inject device with phacoemulsification.The series consisted of eyes with primary open-angle glaucoma(n=52)and pseudoexfoliative glaucoma(n=4).Primary outcome measures included intraocular pressure(IOP)and number of glaucoma medications.Safety outcomes included the need for secondary surgical intervention and the incidence of IOP spikes≥10 mmHg and≥15 mmHg.Results:IOP was reduced by 21%to 14.7±2.9 mmHg(p<0.01)at 6 months postoperative from 18.7±5.8 mmHg at baseline.Preoperatively,the mean number of glaucoma medications was 1.5±0.9 and reduced by 39%to 0.9±1.2(p<0.01)at 6 months.At 6 months,68%of eyes had an IOP≤15 mmHg,increased from 30%at baseline.55%of eyes were medication-free at 6 months,up from 18%at baseline.There were no severe postoperative complications.No eyes underwent an additional glaucoma procedure.Conclusions:Implantation of the iStent inject device with concomitant cataract surgery effectively provides a sustained reduction in IOP with a markedly improved medication burden out to 6 months postoperative.The safety profile is excellent.

关键词： Micro-invasive glaucoma surgery Minimally invasive glaucoma surgery migs Trabecular microbypass stent Open-angle glaucoma

Second-generation trabecular micro-bypass stent implantation: Retrospective analysis after 12- and 24-month follow-up

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Eye and Vision 2020年第1期7卷 1-10页

作者： Raphael Neuhann Tobias Neuhann Neuhann Augentagesklinik MarienplatzMarienplatz 18-1980331 MunichGermany

Background:The study aimed to investigate the 24-month safety and efficacy of implantation of two secondgeneration iStent inject trabecular micro-bypass stents with concomitant cataract surgery.Methods:This consecutive case series included 164 eyes of 109 patients implanted with the iStent inject®device with concomitant cataract surgery.The series was comprised of eyes with primary open-angle glaucoma(n=84),pseudoexfoliation glaucoma(n=42),normal-tension glaucoma(n=18),and ocular hypertension(n=20).All 164 eyes reached 9–14 months of follow-up(“12-month consistent cohort”),with a subset of 88 eyes reaching 21–26 months of follow-up(“24-month consistent cohort”).Performance outcome measures included intraocular pressure(IOP)and number of glaucoma medications.Safety outcomes included intra-or postoperative complications,the need for secondary procedures and corrected distance visual acuity.Comparisons of change in continuous(e.g.,IOP)and categorical(e.g.,proportions of eyes on zero medications)measures between baseline and postoperative times were made with the paired t-test and McNemar’s chi-squared test,respectively.Results:At 12 months postoperatively,IOP was reduced by 25.5%(from 20.0±5.5 mmHg to 14.9±2.0 mmHg;p<0.001);at 24 months postoperatively,IOP was reduced by 26.6%(from 20.3±6.1 mmHg to 14.9±1.9 mmHg;p<0.001).At 12 months postoperatively,mean number of glaucoma medications was reduced by 85.0%(from 2.0±1.0 to 0.3±0.8 medications;p<0.001);at 24 months postoperatively,mean number of medications was reduced by 81.0%(from 2.1±1.1 to 0.4±0.8 medications;p<0.001).After 12 months,96.3%of eyes had an IOP≤18 mmHg and 58.5%of eyes had an IOP≤15 mmHg,with 81.1%of eyes free of any medication,compared to 1.8%of eyes medication-free in the 12-month cohort at baseline.After 24 months,98.9%of eyes had an IOP≤18 mmHg and 53.4%of eyes had an IOP≤15 mmHg,with 72.7%free of medication compared to 1.1%of eyes medication-free in the 24-month cohort at baseline.Overall,a high safety profile was observed with no significant post

关键词： Glaucoma migs IStent inject Trabecular Safety Stent Bypass

In vivo confocal microscopy: qualitative investigation of the conjunctival and corneal surface in open angle glaucomatous patients undergoing the XEN-Gel implant, trabeculectomy or medical therapy

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Eye and Vision 2020年第1期7卷 136-149页

作者： Stefano Baiocchi Cosimo Mazzotta Arianna Sgheri Alessandro Di Maggio Simone Alex Bagaglia Matteo Posarelli Leonardo Ciompi Alessandro Meduri Gian Marco Tosi Department of Medicine Surgery and NeurosciencesOphthalmology UnitPoliclinico Santa Maria alle ScotteUniversity of SienaViale Bracci 8ZIP code 53100 SienaItaly Ophthalmology Unit Department of Biomedical SciencesUniversity of MessinaMessinaItaly

Purpose:Assessing the quality of the ocular surface by in vivo scanning laser confocal microscopy(IVCM)in primary open angle glaucoma(POAG)patients treated by Xen 45 Gel Stent,medical therapy and trabeculectomy.Methods:Retrospective,single-center,single-masked,comparative study including 60 eyes of 30 patients(mean age 61.16±10 years)affected by POAG.Eyes were divided into 3 groups:Group 1 eyes underwent the Xen 45 Gel Stent procedure,Group 2 eyes were under medical therapy,Group 3 eyes were surgically treated by trabeculectomy.All patients underwent HRT II IVCM analysis of cornea,limbus,conjunctiva,sub-tenionian space and sclera.Results:The Xen 45 Gel stent,if properly positioned in the sub-conjunctival space preserves goblet cells and limits ocular surface inflammation.Regular corneal epithelial cells with micro-cysts,and normo-reflective sub-epithelial nerve plexus are documented by IVCM.In sub Tenon’s implants an alternative lamellar intra-scleral filtration is detectable.Combined surgical procedures show a noticeable number of inflammatory cells with rare micro-cysts.Post-trabeculectomy inflammatory reaction is more evident than Xen 45 Gel Stent associated surgical procedures,but less than medical therapy where a conspicuous presence of Langerhans cells,peri-neural infiltrates,marked loss of goblet cells and fibrosis is visible.Conclusion:Ocular surface inflammation was more notable in topical therapy than after trabeculectomy,which itself causes more inflammation than XEN Gel stents.

关键词： Glaucoma Primary open angle glaucoma POAG migs Xen 45 gel stent Ocular surface Inflammation Confocal microscopy IVCM

Long‑term outcomes of two first‑generation trabecular micro‑bypass stents(iStent)with phacoemulsification in primary open‑angle glaucoma:eight‑year results

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Eye and Vision 2021年第1期8卷 431-442页

作者： Ali Salimi Harrison Watt Paul Harasymowycz Department of Ophthalmology Faculty of MedicineMcGill UniversityMontrealQCCanada Montreal Glaucoma Institute MontrealQCCanada Faculty of Medicine McGill UniversityMontrealQCCanada Department of Ophthalmology University of Montreal4135 de RouenMontrealQC H1V1G5Canada

Background:The short-and medium-term outcomes of iStent have been extensively studied;however,only few studies have investigated its long-term outcomes.Here,we assessed the long-term efficacy and safety of two iStents with concomitant cataract surgery in glaucomatous eyes while also evaluating measures of disease stability using visual field and optical coherence tomography(OCT)of the optic nerve and the macula throughout 8 years of follow-up.Methods:This longitudinal,single-center consecutive case series included glaucomatous eyes that underwent implantation of two first-generation trabecular micro-bypass stents(iStent)with concomitant cataract surgery.Eight-year efficacy outcomes included mean intraocular pressure(IOP)and medications,as well as surgical success.Eight-year safety outcomes included best-corrected visual acuity(BCVA),visual field mean deviation(VF-MD),cup-todisc ratio(CDR),retinal nerve fiber layer(RNFL)thickness,ganglion cell-inner plexiform layer(GC-IPL)thickness,and adverse events.Results:A total of 62 eyes with primary open-angle glaucoma(POAG)were included.At 8 years postoperative,IOP reduced by 26%from 19.2±3.9 mmHg preoperatively to 14.2±2.4 mmHg(P<0.001),91.1%of eyes achieved IOP≤18 mmHg(vs.51.6%preoperatively),69.6%of eyes achieved IOP≤15 mmHg(vs.14.5%preoperatively),and 25%of eyes achieved IOP≤12 mmHg(vs.1.6%preoperatively).Medication use decreased by 17.9%from 2.8±1.1 preoperatively to 2.3±1.2(P=0.018).Surgical success was 90%,as six eyes underwent subsequent glaucoma surgeries.Safety measures of BCVA,CDR,RNFL thickness and GC-IPL thickness remained stable through 8 years postoperative.VF-MD remained stable until postoperative year 5 and subsequently progressed according to the natural history of glaucomatous disease.Conclusions:Implantation of two iStents with concomitant cataract surgery is an effective and safe treatment option for surgery-naïve POAG eyes,evidenced by significant IOP and medication reductions,reasonable surgical success,and favorable safety outcomes,throughout the 8-

关键词： Minimally invasive glaucoma surgery migs Trabecular micro-bypass stent iStent Glaucoma Cataract surgery Long-term outcomes

Current review of Excimer laser Trabeculostomy

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Eye and Vision 2020年第1期7卷 232-240页

作者： Georges M.Durr Marc Töteberg-Harms Richard Lewis Antonio Fea Paola Marolo Iqbal Ike K.Ahmed Department of Ophthalmology Universitéde MontréalMontrealQuebecCanada Department of Ophthalmology Centre Hospitalier Universitaire de Montréal(CHUM)1051 Rue SanguinetMontreal H2X 3E4QuebecCanada Department of Ophthalmology University Hospital ZurichZurichSwitzerland Sacramento Eye Consultants SacramentoCaliforniaUSA Struttura Complessa Oculistica Cittàdella Salute e della Scienza di TorinoDipartimento di Scienze ChirurgicheUniversitàdegli Studi di TorinoTorinoItaly Department of Ophthalmology&Vision Sciences University of TorontoTorontoOntarioCanada

Background:Excimer laser trabeculostomy(ELT)is a microinvasive glaucoma surgery(migs)that creates multiple laser channels through the trabecular meshwork using a cold laser system,which minimizes tissue fibrosis and aids in bypassing the main area of resistance to aqueous outflow.The purpose of this review is to evaluate the current body of evidence surrounding ELT in terms of efficacy and review the safety profile of the procedure.Main text:Studies screened had to show clear inclusion and exclusion criteria as well as well-defined outcome measures.PubMed,MEDLINE,EMBASE and the Cochrane Controlled Trial Database were searched.Preferred Reporting Items of Systematic Reviews(PRISMA)guidelines were used to assess for study quality and for any bias.Sixty-four articles were initially identified with 18 meeting preliminary screening criteria.Ultimately,8 studies met inclusion criteria and 2 additional non-referenced publications were also included:1 randomized control trial,4 prospective case series and 5 retrospective studies.Overall studies showed moderate intraocular pressure(IOP)lowering of between 20%and 40%from baseline without medication washout and mostly a decrease in glaucoma medications with few complications.Conclusion:Current literature shows a significant IOP-lowering effect of ELT with a favorable safety-profile in standalone cases or combined with cataract surgery.Limitations to these studies are the lack of controls and washout IOP.Overall,ELT is an attractive migs option that does not require any residual device remaining in the angle.

关键词： Excimer laser trabeculostomy Trabeculotomy ELT Intraocular pressure Glaucoma migs Cataract

XEN® Gel Implant for Glaucoma;Prospective Cohort Study in a High-Volume Department

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Open Journal of Ophthalmology 2023年第2期13卷 208-220页

作者： Hans Wilhelm Meyer Tiril Sandell Department of Ophthalmology Oslo University Hospital Oslo Norway Department of Ophthalmology Vestre Viken Hospital Trust Drammen Norway

Purpose: To evaluate the 3-year efficiency and safety of XEN® 45 gel stent implantation in a heterogenous group of open angle glaucoma patients. Methods: In this prospective, non-randomized observational study we identified patients who had undergone either stand-alone XEN® implantation (XEN solo) or XEN® implantation in combination with phacoemulsification (XEN combi). All patients who had undergone an implantation during the period 01.04.17-31.10.19 at the Department of Ophthalmology, Drammen Hospital, Norway, were asked to participate. Success was defined as IOP between 5 - 18 mmHg and 20% pressure reduction without medications. Qualified success required the same pressure interval and reduction but allowed medications. The procedure was deemed as failure if pressure requirements were not met, vision was reduced to light perception or worse, or if there was a converion to secondary glaucoma surgery. Results: Out of 115 patients and 133 eyes identified, 87 patients and 99 eyes consented to participate. All patients were Caucasians with a mean age of 73.6 years. The study had a mean (range) follow-up of 38.9 (28 - 54) months. The mean medicated baseline (SD) was reduced from 22.6 (7.9) mmHg on 3.2 (1.1) medications to 14.2 (5.6) mmHg on 1.4 (1.6) medications. Success and qualified success were achieved in 22.2% and 21.2%, respectively. Needling was performed in 34 eyes. Conclusion: XEN® 45 gel stent implantation is a safe procedure, offering a significantly lower IOP and number of medications in a subset of patients with open angle glaucoma.

关键词： Long Term Outcome after XEN® Gel Stent Open Angle Glaucoma OAG Pseudoexfoliative Glaucoma Minimally Invasive Glaucoma Surgeries migs

Micro-Invasive Glaucoma Surgery with the iStent inject: Impact on IOP and Medication Burden in a Real-World Setting

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Open Journal of Ophthalmology 2020年第1期10卷 89-98页

作者： Abdulmajeed Alharbi Department of Ophthalmology College of Medicine Qassim University Al-Mulida Qassim KSA

Background: The iStent inject is a Micro-Invasive Glaucoma Surgical (migs) device that has shown to reduce IOP and to be safe for glaucoma patients with fewer complications than regular surgery. Objective: To investigate, up to 15 - 20 months, the efficacy and safety of implantation of two second-generation trabecular microbypass stents in patients with or without prior glaucoma surgery. Methods: Fifty-seven eyes were implanted with the iStent inject. The population was comprised of eyes with primary open-angle glaucoma (n = 51), pseudoexfoliation glaucoma (n = 5) and ocular hypertension (n = 1). Major outcome parameters included IOP, medication needs and corrected distance visual acuity (CDVA). Follow-up time points were one day, 2 - 4 months, 9 - 14 months and 15 - 20 months. Results: The main reason to perform migs was IOP reduction in 68.4%, reduced number of medications due to drug intolerance in 24.6% and reduced medication due to compliance issues in 7.0% of the eyes. IOP decreased by 22.47%, from 19.40 ± 3.83 mmHg preoperatively (preop) to 15.04 ± 1.67 mmHg at 15-20 months postoperatively. IOP reduction was achieved at all follow-up time points (p Conclusions: Insertion of the iStent inject in patients with or without prior glaucoma surgery shows effective and sustained improvements in IOP with no safety concerns.

关键词： iStent inject migs Glaucoma Intraocular Pressure Visual Acuity Medica-tion Need

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migs技术在矮牵牛上的应用

MIGS技术在矮牵牛上的应用

作者：韩垚西南大学

学位级别：硕士

基因沉默技术作为研究基因功能的重要工具之一,在植物分子生物学研究和遗传改良等方面得到广泛应用。自基因沉默现象发现以来,多种基因沉默技术相继建立,包括共抑制、反义RNA、hpRNAi和VIGS等,然而,这些方法都有其局限性。migs沉默技术... 详细信息

基因沉默技术作为研究基因功能的重要工具之一,在植物分子生物学研究和遗传改良等方面得到广泛应用。自基因沉默现象发现以来,多种基因沉默技术相继建立,包括共抑制、反义RNA、hpRNAi和VIGS等,然而,这些方法都有其局限性。migs沉默技术作为一种新的植物基因沉默技术,在基因沉默的高效性方面有了很大改进。migs沉默技术利用拟南芥miR173介导产生一系列tasiRNA, tasiRNA数量巨大,与靶基因互补发挥沉默效应,migs沉默技术调控的效率具有优势。目前,migs沉默技术在矮牵牛上的应用尚未见报道。通过该沉默技术对矮牵牛的八氢番茄红素脱氢酶基因(PDS)和查尔酮合成酶基因(CHS)进行沉默实验,并分析该沉默技术在矮牵牛上沉默效应。以绿色愈伤组织失绿,花颜色发生变化作为标记,表型变化易于观察,可以对基因沉默载体的效果进行快速评价,有利于节省时间,提高工作效率,并且可以为利用migs沉默技术研究矮牵牛相关基因的功能奠定基础。此外,该技术还可以用于对矮牵牛的性状进行改造,为矮牵牛品种改良提供理论和实践指导,因此本实验具有重要的研究意义。获得的结果如下：1、migs沉默技术在矮牵牛上的研究以pGREEN双元载体系统为基础,构建在矮牵牛上应用的migs沉默载体。migs沉默载体包括：miR173基因,带有miR173靶位点的目的基因片段、报告基因。在设计PCR扩增目的基因序列的引物时,将上游引物5’端额外加上与miR173互补的序列,扩增出的目的基因片段就带有了miR173的靶位点,从而在转基因植株内使miR173与连接在目的基因序列5’端的靶序列互补结合行使功能。2、TA克隆在migs沉默载体构建上的应用在拟表达目的片段启动子和终止子序列之间引入两个XcmI酶切位点,得到migs沉默基础表达载体pmigs-T。以pmigs-T为基础构建基因沉默植物表达载体时,可直接将目的基因PCR的扩增产物与XcmI单酶切后的pmigs-T载体相连,选择正向的连接产物,即完成植物表达载体的构建。大大缩短构建过程,节省人力物力。3、矮牵牛PhPDS基因的克隆根据对矮牵牛PDS基因EST序列进行分析,并设计引物。通过RACE技术克隆得到矮牵牛PDS基因的cDNA全长序列,将其命名为PhPDS(GenBank accession number:KP677483)。该基因全长1749bp,编码583个氨基酸。矮牵牛中PDS基因的克隆可以为验证migs沉默技术在矮牵牛中的应用,提供一个直观快捷的内源检测报告基因,并为研究矮牵牛PhPDS基因的进化提供了依据。通过半定量RT-PCR反应检测矮牵牛PhPDS基因在矮牵牛MD不同组织中的表达情况,PhPDS基因有较高的组织表达特异性,在6叶期的茎和叶中表达量最高,根中表达量较低,在成熟期的花中也有少量表达,预测矮牵牛PhPDS基因可能参与植物生长发育的调控。4、植物表达载体的构建选取矮牵牛八氢番茄红素脱氢酶基因(PDS)和查尔酮合成酶基因(CHS)做为靶基因,利用miRl 73诱导的基因沉默(migs)技术体系改变矮牵牛的颜色和花色性状,使转基因植株的表型变化更加容易观察。将目的基因序列连接到表达载体pmigs-T中,构建植物migs沉默载体。分别将重组质粒利用农杆菌介导转入矮牵牛MD品系和地毯品系中,通过一系列的抗性筛选及DNA水平检测,筛选出转基因矮牵牛植株。对转基因矮牵牛进行表型观察发现沉默PhPDS基因的矮牵牛接种大约2周后,开始出现白色的愈伤,沉默CHS基因的矮牵牛转基因植株花瓣褪色,由紫色花变成了白色花,由此证明migs沉默技术体系能够在矮牵牛中应用。5.migs技术对矮牵牛的沉默效应提取表型变化明显的转基因植株RNA,对转录后表达水平检测,结果表明转基因植株中CHS,PDS基因的表达量显著减少;通过RACE技术对CHS,PDS基因的剪切进行检测,结果发现CHS,PDS基因被剪切的位点较多,这与miR173可以介导产生一系列发挥沉默效应的tasiRNA结果一致,对small RNA进行测定,分析沉默的效率。

关键词：矮牵牛 migs PhPDS基因 PhCHS基因基因沉默

基于migs的水稻高效基因沉默体系建立及应用

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基于MIGS的水稻高效基因沉默体系建立及应用

作者：臧黎黎电子科技大学

学位级别：硕士

水稻是世界上最重要的粮食作物之一,全球约二分之一以上的人口以稻米为主食,因此提高水稻的产量和品质尤为重要。随着水稻基因组测序工作的完成,水稻功能基因组学已经成为水稻科研工作的重心,从而发展出多种用于基因功能分析鉴定的新技... 详细信息

水稻是世界上最重要的粮食作物之一,全球约二分之一以上的人口以稻米为主食,因此提高水稻的产量和品质尤为重要。随着水稻基因组测序工作的完成,水稻功能基因组学已经成为水稻科研工作的重心,从而发展出多种用于基因功能分析鉴定的新技术。其中RNA干扰技术(RNA interference,RNAi)作为反向遗传学研究的重要方法已经被广泛应用于各物种的基因功能研究。在植物中有一类称为反式作用小干扰RNAs(trans-acting siRNA,ta-siRNA)的特殊micro-RNAs(miRNAs),它能够诱导产生一系列的小分子干扰RNAs(small interfering RNAs,siRNA)从而导致目的基因沉默。miRNA介导的基因沉默(miRNA-induced gene silencing,migs)技术就是利用了miR173来介导tasiRNA的产生,从而实现目标基因的沉默。migs技术在2012年首次报道能够有效介导拟南芥目标基因的沉默,但是这一高效的基因沉默技术至今仍没有得到有效的应用,migs技术是否可以用于单子叶植物的研究更没有相关报道。因此本研究基于migs工作原理,综合考虑单子叶植物表达形式,构建了miR173和miR173s不同表达模式的migs骨架载体p ZHY930、pZHY931和p ZHY932。首先选取水稻OsPDS为报告基因,通过水稻遗传转化得到转基因白化水稻植株。结果显示三个骨架载体都能介导目标基因的沉默,再生出白化水稻植株,其中p ZH932的沉默效率最高(达到90%),再生产生的白化植株目的基因OsPDS的RNA表达水平明显降低。然后我们又选取了Os ROC5和OsLZAY1两个基因进行migs沉默效应,成功获得了水稻叶片卷叶及分蘖角度增大的水稻突变体植株。再次,我们以p ZHY932为骨架载体,构建了OsPDS和Os ROC5同时沉默,以及OsPDS和OsLZAY1同时沉默的双基因沉默载体,获得了白化卷叶和白化分蘖角度增加的双突变体水稻植株,从而在水稻中建立了migs高效基因沉默体系。基于我们建立的在水稻中高效基因沉默技术——migs技术体系,针对水稻淀粉合成过程中负责合成直链淀粉的OsGBSS基因,构建了migs-OsGBSS载体,并进行了水稻遗传转化。以期利用migs技术沉默OsGBSS基因,对水稻淀粉合成途径进行有效表达调控,获得高支链淀粉。总之,本文基于migs技术体系构建了水稻migs骨架载体。并且利用该骨架载体,选取水稻内源基因,构建了migs-OsPDS、migs-Os ROC5、migs-OsLAZY1、migs双沉默载体以及migs-OsGBSS载体。实现了水稻目标基因的高效沉默,并为migs技术在单子叶植物中的进一步应用提供了有效的理论基础。

关键词：水稻淀粉 tasiRNAs migs

基因叠加和migs技术在水稻淀粉合成途径中的应用

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基因叠加和MIGS技术在水稻淀粉合成途径中的应用

作者：唐爱婷电子科技大学

学位级别：硕士

水稻是世界三大粮食作物之一,全球超过半数的人以稻米为主粮。通过提高水稻单产、改善稻米品质,满足人类在生产生活等方面的多样化需求具有极大的应用价值,水稻种子内淀粉的贮存含量对于水稻的产量有重要作用。淀粉的合成通常是由多种... 详细信息

水稻是世界三大粮食作物之一,全球超过半数的人以稻米为主粮。通过提高水稻单产、改善稻米品质,满足人类在生产生活等方面的多样化需求具有极大的应用价值,水稻种子内淀粉的贮存含量对于水稻的产量有重要作用。淀粉的合成通常是由多种酶参与反应完成,本研究通过应用miRNA诱导的基因沉默技术(miRNA-induced gene silencing, migs)和基因叠加技术(gene stacking, GS)两种不同的基因功能研究方法调控淀粉合成途径中关键基因表达,以期从淀粉组成、淀粉产量两方面来实现水稻产量和品质改良,主要研究内容及结果如下:1、基于前期建立的适用于单子叶植物的高效migs基因沉默体系,我们构建了定向沉默水稻颗粒结合淀粉合成酶基因(GBSSI)的migs-CsGBSSI单基因载体。通过半定量RT-PCR实验检测到OsGBSS1基因在转录水平被抑制表达;对胚乳进行碘染色实验观察发现,野生型呈蓝黑色,转基因胚乳呈红棕色,颜色与糯米染色结果较为接近;此外,在扫描电镜的观察下,转基因胚乳淀粉颗粒为圆形且排列致密,与野生型在形貌上存在一定的区别;利用双波长法测定直链淀粉含量得知,T代胚乳中直链淀粉含量从24.53%下降到16.38%,下调效率达33.2%,与野生型中直链淀粉含量存在极显著性差异,成功实现了 migs技术在淀粉合成途径中的应用。2、.对于GS的多基因共表达体系,我们选取了水稻淀粉合成的重要基因COsAGPL2(pZHY973)、OsAGPS2b(pZHY975),分别构建了两个单基因过表达载体以及一个双基因叠加载体(pZHY976)。转基因实验结果表明,灌浆期20-25DPA的水稻胚乳中OsAGPL2或OsAGPS2b在转录水平都高于野生型,但略低于双基因叠加载体中的单个基因转录表达水平;半定量RT-PCR对水稻进行组织表达模式分析发现,叶片、胚乳中均能检测到OsAGPL2、OsAGPS2b基因的表达,OsAGPL2在根中没有表达;此外,通过蒽酮硫酸法测定水稻胚乳可溶性糖、总淀粉含量,结果表明,转基因植株胚乳的可溶性糖含量均高于野生型且呈极显著差异;相比于野生型,OsAGPL2、OsAGPSb单基因过表达的胚乳总淀粉含量略有提高,双基因叠加的胚乳总淀粉含量最高达到74.31%,增加了约11.7%。此外,3种转基因材料的千粒重指标均高于野生型且存在显著性差异。由此说明,在水稻中建立了适用的淀粉合成相关基因的基因叠加体系。

关键词：水稻淀粉合成 migs 基因叠加