Targeted Genome Editing in Genes and cis- Regulatory Regions Improves Qualitative and Quantitative Traits in Crops

The Clustered Regularly Interspaced Short Palindromic Repeats （CRISPR）-associated protein 9 （CRISPR/Cas9）-based genomeediting system is a revolutionary technology for targeted muta- genesis in molecular biology research and genetic improvement of traits in crops （Cong et al., 2013; Ma et al., 2015, 2016）. Agronomic traits of crops are controlled by major genes and quantitative trait loci （QTL）. Therefore, the CRISPR/Cas9 system can be used to effectively and rapidly produce mutant traits by different strategies （Figure 1A-1C）. The most common application of the targeted editing system in genetic improvement is to knock out completely the functions of target genes, usually by editing site（s） in the coding sequences （CDS） to produce null-allele mutants （Figure 1A）.

Polymorphism in the upstream regulatory region of human papilloma virus type 16 from the cervical cancer biopsies in Xinjiang Uygur women

To investigate the mutations in the upstream regulatory region (URR) of human papillomavirus type 16 (HPV-16) from the cervical cancer biopsies in Xinjiang Uygur women and its relationship to the high incidence of cervical cancer in the southern Xinjiang, the tissue DNA was extracted from the cervical cancer biopsies, and the URR segment of HPV-16 DNA was amplified, sequenced and analyzed. Thereafter, the polymorphism of URR in HPV-16 was then analyzed. It was demonstrated that the positive rate detected for the presence of URR in HPV-16 was 89.47% (17/19). Compared with the previously published sequence in URR of prototype HPV-16, some mutations were detected in the sequence of URR. The mutations in 17 URR fragments of HPV-16 could be divided into 11 patterns (XJU-1 to XJU-11) at nucleic acid level, in which each of XJU-1 and XJU-4 accounted for 23.53% (4/17), and other patterns of mutation accounted for 5.88% (1/17) . In comparison with the URR of prototype HPV-16, the DNA identity of these patterns was 98.50%-99.68% . In these 17 URR fragments, two point mutations occurred at position 7192 (G to T) and position 7520 (G to A) and they appeared to be constant in Xinjiang area. These two mutations were ubiquitous in the Asia-American type and conferred strong infection activity and carcinogenicity of this virus. In addition, the mutations at position 7729 (A to C), position 7843 (A to G) and position 7792 (C to T) could enhance its transcription activity considerably. It is concluded that some mutations occur in URR gene of HPV-16 in the cervical cancer biopsies taken from Uygur women in Xinjiang area, suggesting that certain relationship exists among the mutations in URR of HPV-16, the phylogeny of HPV-16 and the high incidence of cervical cancer in southern part of Xinjiang area.

Association Analysis between a Polymorphism in the 5' Regulatory Region of the IL-6 Gene and Litter Size in Pigs

The aim of this study was to determine the effects of an IL-6 gene polymorphism, discovered in the 5＇ regulatory region, on porcine litter size. An association analysis was performed between the polymorphism and total number born （TNB） and number born alive （NBA） in 421 sows. The polymorphism was at Hpy188I within the 5＇ regulatory region of IL- 6 gene. Three genotypes of AA, AG, and GG were detected in Landrace, and two genotypes, AA and AG, were detected in Yorkshire and Duroc pigs. The A allele was the superior allele in all three breeds, with allele frequencies ranging from 0. 901 to 0.993. The IL-6 genotype was highly significantly associated with TNB and NBA in the third and following parities （ P 〈 0.01 ）, and with total parities （ P 〈 0.05）. In general, the TNB and NBA showed a tendency of GG 〉 AG 〉 AA, indicating that the common allele was the least favorable for litter size. Thus, there is an enormous opportunity to increase litter size if this effect is confirmed in other studies.

Polymorphism in the Regulatory Region of the Aromatase CYP19a Gene in Nile Tilapia

The aim of this study was to evaluate the polymorphism in a portion of the gene regulatory region for ovarian aromatase （CYP19a） in three strains of Tilapia, Oreochomis niloticus （Linnaeus） （GIFT--Genetically Improved Farmed Tilapia, Chitralada and Supreme）. A total of 90 animals per strain of Tilapia, Oreochromis niloticus （Linnaeus） were analysed. After DNA extraction, samples were subjected to PCR using primers designed to flank the region of interest encompassing the sites of transcription （WT1-KTS and SRY）. Samples were analyzed by PCR-SSCP and subsequently sequenced. Three polymorphisms were identified in this region, resulting in two different sequences, in the GIFT strain while no polymorphism was found in both Supreme and Chitralada strains. At the position - 1178 the substitution of a guanine for a cytosine, at the - 1081 the exchange of guanine for adenine and at the position -1 138 we found a SNP, possible site of heterozygosity. Even with polymorphisms in the target study area, when taking the three strains into account, one can assume that the portion of the regulatory region of the ovarian aromatase gene in the Supreme strain and Chitralada does not show polymorphism.

Transfection and expression of exogenous gene in laying hens oviduct in vitro and in vivo

To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3.0 kilobases (kb) of the 5'-flanking sequence and 3.0 kb of the 3'-flanking sequence of the chicken ovalbumin gene. Jellyfish green fluorescence protein (EGFP) reporter gene and bacterial LacZ reporter gene were respectively inserted into the downstream of the 5'-regulatory region. The recombinants were named as pOVEGFP and pOVLacZ. Two transfer systems, in vitro and in vivo, were used to verify the function of the vector. In vitro, the plasmid DNA pOVEGFP and pEGFP-N1 were transfected respectively by the polyethyle-neimine procedure into the primary chicken oviduct epithelium (PCOE) and fibroblasts cells isolated from laying hens. In vivo, the recombinant vector pOVLacZ was injected into egg-laying hens via wing vein and the tissues were collected for RT-PCR analysis. The results showed that expression of pEGFP-Nl was achieved at low level in oviduct epithelial cells and at high level in fibroblasts, but that the recombinant vector was not expressed in both cells. RT-PCR analysis showed that the LacZ gene was transcribed in the oviduct, but not in the heart, liver, kidney and spleen of the injected hens. Accordingly, the β-galactosidase activity was only detected in the oviduct magnum (116.7 mU/ml) and eggs (16.47 mU/ml). These results indicated that the cloned regulation regions of chicken ovalbumin gene could drive exogenous gene expression specifically in the oviducts of hens. In vivo gene injection via wing vein may serve as a rapid production system of recombinant proteins in chicken eggs. In addition, the cultured primary oviduct cells from laying hens were not efficient temporary expression systems for analyzing the function of regulating elements of ovalbumin gene.

Waxy allele diversity in waxy maize landraces of Yunnan Province, China

Waxy maize is one of the main fresh-eating maize types,and a mutation of the waxy gene causes the waxy character of maize grains.China is rich in waxy maize landraces,and Yunnan and its surrounding areas,are the place of origin and genetic diversity center of Chinese waxy maize.The six known waxy alleles of Chinese waxy maize are wx-D7,wx-D10,wx-Cin4,wx-124,wx-Reina,and wx-Xuanwei.The mutation sites of these alleles all occur in the coding region of the waxy gene,however,the mechanism by which the waxy characteristic is caused by the mutation in the regulatory region has only been reported rarely in maize.In this study,405 waxy maize landraces from Yunnan were used as materials to identify the insertion and deletion of a large sequence fragment in the upstream~3.5 kb regulatory region of the waxy gene by molecular marker detection.Three different waxy alleles were identifed in this study:wx-PIF/Harbinger,wx-hAT and wxElote2.These three types of mutations all represented transposons inserted into the regulatory region of the waxy gene.Wx-PIF/Harbinger was a 304-bp MITE class transposon insertion belonging to the PIF/Harbinger family,while wx-hAT was a 560-bp MITE class transposon insertion belonging to the hAT family,and wx-Elote2 was a 6560-bp LTR-like transposon insertion.In this study,the alleles were identifed for more than 70%of the waxy maize landraces in Yunnan,which provids a basis for the utilization of these waxy maize landraces.

Complete Genomic Sequence Analysis of a New SV40 Isolate

从恒河猴孤立的一个新 SV40 种类(SV-IMB ) 的染色体完全被定序并且与其它相比孤立。结果证明整个染色体包含 5246bp，并且 SV-IMB 的 aver 年龄身份作为与另外的 SV40 相比是 98.1% 孤立。它的规章的区域由完全的 enhancer 和有缺点的 e enhancer 组成。氨基酸变化在大 T 抗原(标签) 和 VP1 区域发生了到某程度。调查结果证明 SV-IMB 是新 SV40 孤立。关键词 SV40 - 标签 - 规章的区域 CLC 数字 S852.65 基础条款：国家自然科学成立(30570081 )

Cloning and analysing of 5' flanking region of Xenopus organizer gene noggin

Xenopus organizer specific gene Noggin possessesnearly all the characterestic properties of the action of or-ganizer to specify the embryonic body axis. To analyzehow the maternal inherited factors cofltrol its expressionpattern, we cloned the 5’ regulatory region of noggn gene.The 1.5 kb upstream sequense could direct reporter geneto express in vivo and data from deletion analysis indi-cated that a 229 base pair fragmet is essential for acti-vating noggn expression. We further demonstrated thatthe response elements within this regulatory region wereindeed under the control of growth factor activin and Wntsignaling pathway components.

Mitigating cadmium accumulation in rice without compromising growth via modifying the regulatory region of OsNRAMP5

Cadmium(Cd)intake poses a significant health risk to humans,and the contamination of rice grains with Cd is a major concern in regions where rice is a staple food.Although the knockout of OsNRAMP5,which encodes a key transporter responsible for Cd and manganese(Mn)uptake,can significantly reduce Cd accumulation in rice grains,recent studies have revealed that this knockout adversely affects plant growth,grain yield,and increases vulnerability to abiotic and biotic stresses due to reduced Mn accumulation.In this study,we employed CRISPR/Cas9 technology to modify the regulatory region of OsNRAMP5 with the aim of reducing Cd accumulation in rice grains.Our findings demonstrate that mutations in the regulatory region of OsNRAMP5 do not impact its expression pattern but result in a reduction in translation.The decreased translation of OsNRAMP5 effectively decreases grain Cd accumulation while leaving Mn accumulation and important agronomic traits,including yield,unaffected.Thus,our study presents a practical and viable strategy for reducing Cd accumulation in rice grains without compromising Mn accumulation or overall rice production.