Genome-wide analysis of brassinosteroid responsive small RNAs in Arabidopsis thaliana

박소영,Jae‑Han Choi,Dong‑Ha Oh,John C. Johnson,Maheshi Dassanayake,Dong‑Hoon Jeong,Man‑Ho Oh 한국유전학회 2020 Genes & Genomics Vol.42 No.8

Background Brassinosteroids (BRs) are a class of phytohormones with important roles in regulating physiological and developmental processes. Small RNAs, including small interfering RNAs and microRNAs (miRNAs), are non-protein coding RNAs that regulate gene expression at the transcriptional and post-transcriptional levels. However, the roles of small RNAs in BR response have not been studied well. Objective In this study, we aimed to identify BR-responsive small RNA clusters and miRNAs in Arabidopsis. In addition, the effect of BR-responsive small RNAs on their transcripts and target genes were examined. Methods Small RNA libraries were constructed from control and epibrassinolide-treated seedlings expressing wild-type BRI1-Flag protein under its native promoter in the bri1-5 mutant. After sequencing the small RNA libraries, differentially expressed small RNA clusters were identified by examining the expression levels of small RNAs in 100-nt bins of the Arabidopsis genome. To identify the BR-responsive miRNAs, the expression levels of all the annotated mature miRNAs, registered in miRBase, were analyzed. Previously published RNA-seq data were utilized to monitor the BR-responsive expression patterns of differentially expressed small RNA clusters and miRNA target genes. Results In results, 38 BR-responsive small RNA clusters, including 30 down-regulated and eight up-regulated clusters, were identified. These differentially expressed small RNA clusters were from miRNA loci, transposons, protein-coding genes, pseudogenes and others. Of these, a transgene, BRI1, accumulates small RNAs, which are not found in the wild type. Small RNAs in this transgene are up-regulated by BRs while BRI1 mRNA is down-regulated by BRs. By analyzing the expression patterns of mature miRNAs, we have identified BR-repressed miR398a-5p and BR-induced miR156g. Although miR398a5p is down-regulated by BRs, its predicted targets were not responsive to BRs. However, SPL3, a target of BR-inducible miR156g, is down-regulated by BRs. Conclusion BR-responsive small RNAs and miRNAs identified in this study will provide an insight into the role of small RNAs in BR responses in plants. Especially, we suggest that miR156g/SPL3 module might play a role in BR-mediated growth and development in Arabidopsis.

Identification and analysis of microRNAs in Candida albicans

Jin-Hyun Cho(조진현),Heon-Jin Lee(이헌진) 한국생명과학회 2017 생명과학회지 Vol.27 No.12

Candida albicans에 의한 구강 감염(캔디다증)은 구강 점막에 빈번하게 발생하며 잘 알려진 질병이다. 구강 캔디 다증은 생명을 위협하는 정도의 곰팡이 감염증은 아니나, 특정상황에서 개인에게 심각한 위험을 초래할 수도 있다. 마이크로 RNA는 세포 내에서 다른 타겟 유전자를 저해하는 작은 크기의 RNA 분자이며 단백질을 코딩하지는 않고 번역과정을 억제하는 조절자로서의 역할을 하고 있다. 본 연구는 C. albicans의 마이크로RNA를 처음으로 동정하고 그러한 마이크로RNA가 지닌 기능을 조사하기 위함이다. 이를 위하여 C. albicans의 small RNA를 차세대 염기분석법을 통하여 분석하고 그러한 RNA들의 2차 구조를 생물정보학적 방법으로 조사하였다. 분석한 small RNA들은 마이크로 RNA라고 불리울 수 있는 특징들을 가지고 있었으며, 특별히 높게 발현되고 있는 두개의 마이크로 RNA 정도 크기의 RNA가 CBP1 유전자의 3’ 말단 비번역구역(UTR)에서 반대방향으로 발현하는 것을 밝혀내었다. 우리는 이러한 C. albicans의 RNA가 CBP1 유전자를 타겟으로 하여 조절하는지 알아보기 위해 RNA를 인위적으로 합성한 후 세포 내로 주입하고, 형광형미경으로 도입 사실을 확인하였다. 하지만 4시간과 8시간 후에 CBP1의 발현 변화는 관찰되지 않았다. 따라서, 이러한 결과는 C. albicans가 마이크로RNA에 의한 RNA 간섭(RNAi) 작용이 다른 진핵세포와는 다르게 작용하는 것을 알 수 있다. Oral infection due to Candida albicans is a widely recognized and frequent cause of superficial infections of the oral mucosa (oral candidiasis). Although oral candidiasis is not a life-threatening fungemia, it can cause severe problems in individuals under certain conditions. MicroRNAs (miRNAs) are noncoding, small RNA molecules, which regulate the expression of other genes by inhibiting the translation of target mRNAs. The present study was designed to identify miRNAs in C. albicans and determine their possible roles in this organism. miRNA-sized small RNAs (msRNAs) were cloned in C. albicans by deep sequencing, and their secondary structures were analyzed. All the cloned msRNAs satisfied conditions required to qualify them as miRNAs. Bioinformatics analysis revealed that two of the most highly expressed C. albicans msRNAs, Ca-363 and Ca-2019, were located in the 3" untranslated region of the corticosteroid-binding protein 1 (CBP1) gene in a reverse orientation. miRNA mimics were transformed into C. albicans to investigate their RNA-inhibitory functions. RNA oligonucleotide-transformed C. albicans was then observed by fluorescent microscopy. Quantitative PCR analysis showed that these msRNAs did not inhibit CBP1 gene expression 4 hr and 8 hr after ectopic miRNA transformation. These results suggest that msRNAs in C. albicans possess an miRNA-triggered RNA interference gene-silencing function, which is distinct from that exhibited by other eukaryotic systems.

장기이식에서의 RNA간섭 치료법의 전망

김재영 대한이식학회 2015 Korean Journal of Transplantation Vol.29 No.3

RNA간섭(RNA interference, RNAi)은 작은 크기의RNA가 염기서열 특이적으로 유전자 발현을 조절하는 세포 내에 정상적으로 존재하는 현상을 말한다(1-3). RNA 간섭에 관여하는 작은 RNA는 크게 두 가지가 있는데, 하나는 miRNA (microRNA)이고, 다른 하나는 siRNA (small interfering RNA)이다(4,5). miRNA는 원래 세포 내 유전자로부터 발현되는 전사체로써 전사후유전자침묵(posttranscriptional gene silencing)을 매개하며, siRNA는 외부로부터 세포 내로 도입된 이중가닥 RNA (dsRNA)로 자신과상보적인 염기서열을 지닌 mRNA를 절단한다(4,5). RNA 간섭의 작용방식이 표적 mRNA의 염기서열에 고도로 특이적이기 때문에 염기서열만 안다면 이론적으로 원하는어떠한 유전자의 발현도 조절 가능하다. 이러한 이유로, 다양한 의생명 연구에 광범위하게 이용되고 있으며, 최근에는 전통적인 약물치료의 대안으로써 다양한 질환에 RNA 간섭 치료법을 적용하려는 시도가 늘고 있다. 본 논문에서는 RNA간섭의 원리, 적용 및 장기이식에서의 RNA간섭치료연구의 예들을 살펴 봄으로써, 장기이식 분야에서RNA간섭 치료법의 임상적용에 대해 전망해보고자 한다. RNA interference (RNAi) is a normal cellular process in which small RNAs control gene expression. siRNAs introduced into cells suppress gene expression through their recognition and cleavage of cognate mRNAs in a sequence specific manner. Due to its highly specific mode of action, RNAi has recently been tested for treatment or prevention of various diseases including organ transplantation as well as basic biomedical research. However, to achieve clinical success, there are some important issues that should be fully validated. First, siRNAs should be properly designed to avoid off-target effects. Second, siRNAs must be modified so as not to induce innate immune responses. Third, selective delivery of siRNA into desired organs or tissues is required. Despite such prerequisites, siRNAs are thought to be superior to traditional small molecule drug in terms of new drug development. In addition, in case of heart and islet transplantation which probably requires preservation of organs or cultivation of tissues for a while, siRNAs can be added to preserving solution or medium to control target gene expression during this period. In many research studies, mediators of innate immune response, inflammation, and cell death have been tested for alleviation of tissue injury and immune rejection after transplantation as potent targets of RNAi. We suggest that elucidation of exact mechanisms for tissue injury and immune rejection and subsequent selection and validation of target of RNAi in future studies might be helpful in enabling RNAi-based therapy in clinical organ transplantation to become a reality.

RNA Therapy: Current Status and Future Potential

김영국 전남대학교 의과학연구소 2020 전남의대학술지 Vol.56 No.2

Recent studies identified diverse RNAs including noncoding RNAs and their various action mechanisms in the cells. These RNAs regulate a variety of cellular pathways and are therefore expected to be important targets for the treatment of human diseases. Along with their extensive functional studies, RNA-based therapeutic techniques have developed considerably in recent years. After years of research and various trial and error, antisense RNAs and small interfering RNAs-based drugs have been developed and are now being used in the clinic. In addition, active research is ongoing to develop drugs based on RNA aptamer and messenger RNA. Along with the development of these RNA-based drugs, diverse strategies have been developed to transport RNA drugs into the cells efficiently. RNA therapy has many advantages over existing small molecule or monoclonal antibody-based therapies, including its potential to target all genes in the cells. This review will introduce the history of RNA therapy, and explain the basic concepts of RNA therapy and RNA-based drugs on the market or clinical trials. In addition, the future potential of RNA therapy will be discussed.

자연적으로 유발된 고혈압 백서에서 안지오텐신 전환효소를 억제하는 Small Hairpin RNA 투여 효과

홍영미,이혜련,김관창 대한고혈압학회 2012 Clinical Hypertension Vol.18 No.3

Background: Interfering RNA (iRNA) represents a recent breakthrough in effective blocking of the target genes in mammalian cells. Angiotensin-converting enzyme (ACE) has been shown to play an important role in the pathogenesis of hypertension. The purposes of this study were to investigate the effects on blood pressure, myocardial hypertrophy and gene expressions of iRNA targeting ACE. Methods: Twelve week old male Wistar-Kyoto rats were grouped as follows: control group (C group), spontaneously hypertensive rat (SHR) group (H group), and ACE-iRNA group (A group) in which SHR was treated with recombinant lentiviral vectors carrying small hairpin RNA targeting ACE. Reverse transcriptionpolymerase chain reaction and western blot analysis of ACE, endothelin (ET)-1, angiotensin (AT) II receptor type 1A,neutrophil cytosolic factor, caspase 3, Bax, and Bcl-2 were performed in the heart tissues. Serum AT, ACE, and high sensitive-C reactive protein were estimated. Results: Systolic blood pressure was significantly decreased in the A group compared with the H group in weeks 3 and 5. Serum AT level was significantly lower on day 1, weeks 3 and 5 after ACE-iRNA treatment. ACE protein contents were significantly lower after ACE-iRNA treatment in week 5. ET-1 and Bcl-2protein contents were significantly lower after ACE-iRNA treatment in weeks 3 and 5. Bax protein contents were significantly lower after ACE-iRNA treatment in week 3. Conclusions: Recombinant lentiviral vectors carrying shRNA targeting ACE prevented hypertension. Serum AT and gene expressions such as ACE, ET-1, Bax, and Bcl-2 were significantly decreased after ACE-iRNA treatment. 연구배경: RNA interference는 이중 가닥의 RNA (double-stranded RNA)가 dicer에 의해 절단되어 생성되는 21-25 뉴클레오타이드 크기의 작은 RNA조각으로 상보적인 서열을 갖는 mRNA에 특이적으로 결합하여 단백질 발현을 억제하는 것으로 밝혀졌다. 본 연구에서는 자연적으로 유발된 고혈압 쥐(spontaneously hypertensive rat, SHR) 모델에서 안지오텐신 전환효소(angiotensinconverting enzyme, ACE)에 대한 small hairpin RNA (shRNA)가 주입된 렌티바이러스 벡터를 투여하여 혈역학적, 해부학적 변화를 알아보고, 이와 같은 조절 효과가조직학적 변화 또는 유전자 수준에서 어느 시기에 어느정도 일어나는지를 규명하고자 하였다. 방법: 12주의 자성 성숙 Wistar-Kyoto 백서를 대조군으로 SHR을 고혈압군(H군)으로 shRNA가 주입된 렌티바이러스 벡터를 투여한 치료군을 ACE-interfering RNA (iRNA) (A군)으로 하였다. shRNA 투여는 렌티바이러스에 형질주입된 상태로 1 mL를 실험 시작일에 SHR의 꼬리에 정맥투여하였다. 대조군 및 실험군 모두 21일, 35일에 도살하였다. 혈청 알도스테론, ACE, 안지오텐신치를측정하였고, reverse transcription-polymerase chain reaction과Western blot분석을 이용하여 endothelin (ET)-1,ACE, angiotensin II receptor (AT II R) 1A, neutrophil cytosolic factor (NCF), caspase-3, Bax, Bcl-2 유전자 발현을 측정하였다. 결과: 혈압은 H군에서 유의하게 증가하였고 A군에서유의하게 감소하였다. 혈청 안지오텐신치는 H군에서 유의하게 증가하였고, A군에서 유의하게 감소하였다. ACE 농도는 세 군 간에 유의한 차이가 없었다. ACE 농도는 5주에, ET-1와 Bcl-2 단백질 농도는 ACE-iRNA 치료 후 3주, 5주째 유의하게 감소하였디. Bax 단백질 농도는ACE-iRNA 치료 후 3주에 유의하게 감소하였다. AT II R, NCF, caspase-3 단백질 농도는 ACE-iRNA 치료 후 유의한 감소가 없었다. 결론: ACE에 대한 shRNA가 주입된 렌티바이러스 벡터는 항고혈압 효과가 뚜렷하였고, ACE, ET-1, Bax,Bcl-2 유전자 발현의 감소를 초래하였다. 향후 shRNA 주입 양을 증량시킨 연구가 추후에 이루어져야 할 것으로생각한다.

Similarities and differences between 6S RNAs from Bradyrhizobium japonicum and Sinorhizobium meliloti

Olga Y. Burenina,Daria A. Elkina,Anzhela Y. Migur,Tatiana S. Oretskaya,Elena Evguenieva-Hackenberg,Roland K. Hartmann,Elena A. Kubareva 한국미생물학회 2020 The journal of microbiology Vol.58 No.11

6S RNA, a conserved and abundant small non-coding RNA found in most bacteria, regulates gene expression by inhibiting RNA polymerase (RNAP) holoenzyme. 6S RNAs from α-proteobacteria have been studied poorly so far. Here, we present a first in-depth analysis of 6S RNAs from two α-proteobacteria species, Bradyrhizobium japonicum and Sinorhizobium meliloti. Although both belong to the order Rhizobiales and are typical nitrogen-fixing symbionts of legumes, their 6S RNA expression profiles were found to differ: B. japonicum 6S RNA accumulated in the stationary phase, thus being reminiscent of Escherichia coli 6S RNA, whereas S. meliloti 6S RNA level peaked at the transition to the stationary phase, similarly to Rhodobacter sphaeroides 6S RNA. We demonstrated in vitro that both RNAs have hallmarks of 6S RNAs: they bind to the σ70-type RNAP holoenzyme and serve as templates for de novo transcription of so-called product RNAs (pRNAs) ranging in length from ~13 to 24 nucleotides, with further evidence of the synthesis of even longer pRNAs. Likewise, stably bound pRNAs were found to rearrange the 6S RNA structure to induce its dissociation from RNAP. Compared with B. japonicum 6S RNA, considerable conformational heterogeneity was observed for S. meliloti 6S RNA and its complexes with pRNAs, even though the two 6S RNAs share ~75% sequence identity. Overall, our findings suggest that the two rhizobial 6S RNAs have diverged with respect to their regulatory impact on gene expression throughout the bacterial life cycle.

식물의 긴비암호화 RNA들의 생물학적 기능

김지혜(Jee Hye Kim),허재복(Jae Bok Heo) 한국생명과학회 2016 생명과학회지 Vol.26 No.9

차세대 염기서열 분석기술의 발달로 대량의 전사수준의 단위체들이 발견되었는데, 이것들 중 대부분의 전사체들은 비암호화 RNA들이다. 이들 중 긴 비암호화 RNA (lncRNA)들은 200개 이상의 뉴클레오티드를 가지며 단백질로 번역이 되지 않는 기능적 RNA 분자들이다. 식물의 lncRNA들은 RNA Pol II, Pol III, Pol IV, Pol V에 의해 전사체가 만들어지고, 전사 후 이들 lncRNA들은 추가적인 splicing과 polyadenylation 과정이 일어난다. 식물의 lncRNA들은 그 발현수준이 매우 낮고, 조직 특이적으로 발현 되지만, 이들 lncRNA들은 외부자극에 의해 강한 발현이 유도된다. 환경스트레스를 포함한 각기 다른 외부자극에 의해 많은 식물 lncRNA들의 발현이 유도되었기 때문에 이들 lncRNA들은 식물의 다양한 생물학적 기능과 식물의 생장발달 과정에 있어 새로운 조절 인자로 고려되고 있다. 특히 후성유전학적인 유전자 억제, 크로마틴 변형, 타겟모방, 광형태형성, 단백질 재배치, 환경스트레스 반응, 병원균 감염등에 관련하여 기능을 한다. 또한 어떤 lncRNA들은 short RNA들의 전구체로 역할도 한다. 최근 식물에서 많은 lncRNA들이 분리 동정되었지만, 이들 lncRNA들의 생리학적인 기능에 대한 현재의 이해는 여전히 제한적이고, 이들의 세부적인 조절 메커니즘 연구는 지속적으로 이루어져야 할 것이다. 이 총설에서는 식물 lncRNA들의 생합성 및 조절 메커니즘과 현재까지 밝혀진 분자수준에서의 중요한 기능들을 요약 정리하였다. With the development of next generation sequencing (NGS), large numbers of transcriptional molecules have been discovered. Most transcripts are non -coding RNAs (ncRNAs). Among them, long non-coding RNAs (lncRNAs) with more than 200 nucleotides represent functional RNA molecule that will not be translated into protein. In plants, lncRNAs are transcribed by RNA polymerase II (Pol II) or Pol III, Pol VI and Pol V. After transcription of these lncRNAs, more RNA processing mechanisms such as splicing and polyadenylation occurs. The expression of plant lncRNAs is very low and is tissue specific. However, these lncRNAs are strongly induced by specific external stimuli. Because different external stimuli including environmental stresses induce a large number of plant lncRNAs, these lncRNAs have been gradually considered as new regulatory factors of various biological and development processes such as epigenetic repression, chromatin modification, target mimicry, photomorphogenesis, protein relocalization, environmental stress response, pathogen infection in plants. Moreover, some lncRNAs act as precursor of short RNAs. Although a large number of lncRNAs have been predicted and identified in plants, our current understanding of the biological function of these lncRNAs is still limited and their detailed regulatory mechanisms should be elucidated continuously. Here, we reviewed the biogenesis and regulation mechanisms of lncRNAs and summarized the molecular functions unraveled in plants.

다시마 (Laminaria japonicus) Alginate의 가열가수분해에 따른 물리$\cdot$화학적 및 생물학적 특성에 관한 연구 5. 랫드의 체중, 장기, 췌장과 소장의 성분 및 소장융모의 미세구조에 미치는 저분자 Alginate의 영향

김육용,조영제,KIM Yuck-Yong,CHO Young-Je 한국수산과학회 2001 한국수산과학회지 Vol.34 No.1

Alginate를 가열에 의해 저분자화하여 저분자화에 따른 소화생리 특성을 검토하기 위해, 랫드에 저분자 alginate인 HAG-10,HAG-50, HAG-100 및 alginate를 장기간 섭취시켰을 때, 체중, 각종 장기의 무게와 길이 및 췌장과 소장의 소화효소활성과 단백질, DNA 및 RNA 함량을 측정하였고 소장응모의 미세구조의 변화를 관찰하였다. 랫드의 체중은 $5\%$와 $10\%$ HAG-50 및 $10\%$ alginate에서 체중의 증가가 유의적으로 현저히 억제되었다. 간장의 무게는 유의적인 차이가 없었고, 위의 무게와 길이는 $10\%$ HAG-10, $1\%$, $5\%$ 및$10\%$ HAG-50, $1\%$와 $5\%$ HAG-100 그리고 $5\%$ alginate에서 증가하는 경향을 보였으며, 췌장의 무게와 길이는 모두 약간 증가하였다. 소장과 맹장의 무게와 길이는 $1\%$와 $5\%$ HAG-10을 제외하고 모두 증가하였으나 대장의 무게와 길이는 전반적으로 감소하였다. 췌장의 amylase 활성은 유의적인 차이는 보이지 않았고, lipase 활성은 HAG-50에서 약간 저하하였으며, protease 활성은 $1\%$ HAG-10만 제외하고 유의적으로 저하하였다. 췌장의 단백질 함량은 모두 증가하였으나, DNA와 RNA 함량은 유의 적 인 차이가 없었다. 소장의 단백질 함량은 $5\%$와 $10\%$ HAG-50에서 그리고DNA는 $5\%$ HAG-50에서 가장 높은 함량을 보였으며, RNA는 전반적으로 증가하였다 소장융모의 미세구조는 HAG-50에서 주름이 많고 표면적이 넓은 잎사귀 모양의 응모세포와 돌림주름 및 Boblet cell이 현저히 발달되어 있었다. To examine functionality of depolymerized alginate obtained by hydrolysis of alginate through a heating process at $121^{\circ}C$ on gastrointestinal physiology, the changes of body weight, organ weight and length, pancreatic and small intestinal composition, and light microscopy (LM) observation of small intestinal microvilli's appearances were checked in the rats. Rats were fed diets containing $1\%, 5\%, and 10\%$ of each depolymerized alginate (HAG-10, HAG-50, HAG-100) and alginate for 35 days, The feeding of 5 and $10\%$ HAG-50 and $10\%$ alginate diets for 35 days significantly depressed the body weight gain, but increased the length and weight of the small intestine and cecum in rats (p<0.01). Pancreatic protease activity was decreased significantly (p<0.01) in all groups except lo/o of HAG-10 diets, but the protein content increased in all groups, However, pancreatic amylase and lipase activities as well as DNA and RNA content were not significantly different. The small intestinal protein and the DNA content were the highest in diets fed $5\%$ HAG-50; RNA content increased significantly (p<0.01) in all groups except in the fiber-free diets. Light microscopy (LM) observation showed growth of small intestinal microvilli with numerous ridges; the multiplication of the convolution goblet cells in rats fed with diets containing $5\%$ of HAG-50 were more than others group.

RNA Interference-Mediated Simultaneous Silencing of Four Genes Using Cross-Shaped RNA

이태연,이동기,장찬일,이두영,홍선우,신찬석,Chiang J. Li,김소연,Dirk Haussecker 한국분자세포생물학회 2013 Molecules and cells Vol.35 No.4

The structural flexibility of RNA interference (RNAi)-trig-gering nucleic acids suggests that the design of uncon-ventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA trig-gers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.

Small non-coding RNA를 발현하는 형질전환 벼의 환경위해성 평가 방법

김민철,진병준,전현진,조현민,이수현,최철우,정욱헌,백동원,한창덕 한국식물생명공학회 2019 JOURNAL OF PLANT BIOTECHNOLOGY Vol.46 No.3

Since the RNA interference (RNAi) had been discovered in many organisms, small non-coding RNAmediated gene silencing technology, including RNAi have been widely applied to analysis of gene function, as well as crop improvement. Despite the usefulness of RNAi technology, RNAi transgenic crops have various potential environmental risks, including off-target and non-target effects. In this study, we developed methods that can be effectively applied to environmental risk assessment of RNAi transgenic crops and verified these methods in 35S::dsRNAi_eGFP rice transgenic plant we generated. Off-target genes, which can be non-specifically suppressed by the expression of dsRNAi_eGFP, were predicted by using the published web tool, pssRNAit, and verified by comparing their expressions between wild-type (WT) and 35S::dsRNAi_eGFP transgenic rice. Also, we verified the non-target effects of the 35S:: dsRNAi_eGFP plant by evaluating horizontal and vertical transfer of small interfering RNAs (siRNAs) produced in the 35S::dsRNAi_eGFP plant into neighboring WT rice and rhizosphere microorganisms, respectively. Our results suggested that the methods we developed, could be widely applied to various RNAi transgenic crops for their environmental risk assessment.