The Application of Nicotiana benthamiana as a Transient Expression Host to Clone the Coding Sequences of Plant Genes

Coding sequences (CDS) are commonly used for transient gene expression, in yeast two-hybrid screening, to verify protein interactions and in prokaryotic gene expression studies. CDS are most commonly obtained using complementary DNA (cDNA) derived from messenger RNA (mRNA) extracted from plant tissues and generated by reverse transcription. However, some CDS are difficult to acquire through this process as they are expressed at extremely low levels or have specific spatial and/or temporal expression patterns in vivo. These challenges require the development of alternative CDS cloning technologies. In this study, we found that the genomic intron-containing gene coding sequences (gDNA) from Arabidopsis thaliana, Oryza sativa, Brassica napus, and Glycine max can be correctly transcribed and spliced into mRNA in Nicotiana benthamiana. In contrast, gDNAs from Triticum aestivum and Sorghum bicolor did not function correctly. In transient expression experiments, the target DNA sequence is driven by a constitutive promoter. Theoretically, a sufficient amount of mRNA can be extracted from the N. benthamiana leaves, making it conducive to the cloning of CDS target genes. Our data demonstrate that N. benthamiana can be used as an effective host for the cloning CDS of plant genes.

Crude enzyme immobilization‑based cell‑free system for efficient N‑acetylneuraminic acid biosynthesis aided by N‑terminal coding sequence screening

N-acetylneuraminic acid(NeuAc)is an important nutrient that plays a key role in brain development in infants NeuAc is mainly produced by extraction from natural resources such as edible birds’s nests,crucian eggs,caviar and human breast milk.The extraction process is complicated,resulting in the disadvantages of low NeuAc content and low recovery rate.In this study,a crude enzyme immobilization-based cell-free system(CEICFS)was developed for efficient NeuAc biosynthesis.First,N-terminal coding sequences that improved the expression levels of N-acetylglucosamine-2-epimerase(AGE)and N-acetylneuraminic acid aldolase(NanA)were obtained by high-throughput screening.And these sequences resulted in up to 1.5-fold(1.2-fold)increase in AGE(NanA)enzyme levels.And then,a CEICFS for NeuAc biosynthesis was proposed by directly immobilizing crude enzyme containing AGE and NanA on amino resin.Subsequently,NeuAc production from GlcNAc using CEICFS in one reactor was carried out,resulting 68 g/L of NeuAc and the highest productivity of 6.8 g/L/h.Further,the enzyme activity was still higher than 75%after five repeated uses.The functional properties of CEICFS were studied and compared to those of the free enzyme,immobilization can extend the application of enzyme to some harsh environments,such as low temperature and acidic environment.Therefore,CEICFS with excellent heat resistance,storage stability and reusability exhibit great potential for industrial application.

Landscape of Sequence Variations in Homologous Copies of FAD2 and FAD3 in Rapeseed(Brassica napus L.)Germplasm with High/Low Linolenic Acid Trait

Genetic manipulation(either restraint or enhancement)of the biosynthesis pathway ofα-linolenic acid(ALA)in seed oil is an important goal in Brassica napus breeding.B.napus is a tetraploid plant whose genome often har-bors four and six homologous copies,respectively,of the two fatty acid desaturases FAD2 and FAD3,which con-trol the last two steps of ALA biosynthesis during seed oil accumulation.In this study,we compared their promoters,coding sequences,and expression levels in three high-ALA inbred lines 2006L,R8Q10,and YH25005,a low-ALA line A28,a low-ALA/high-oleic-acid accession SW,and the wildtype ZS11.The expression levels of most FAD2 and FAD3 homologs in the three high-ALA accessions were higher than those in ZS11 and much higher than those in A28 and SW.The three high-ALA accessions shared similar sequences with the pro-moters and CDSs of BnFAD3.C4 and BnFAD3.A3.In A28 and SW,substitution of three amino acid residues in BnFAD2.A5 and BnFAD2.C5,an absence of BnFAD2.C1 locus,and a 549 bp long deletion on the BnFAD3.A3 promoter were detected.The profile of BnFAD2 mutation in the two low-ALA accessions A28 and SW is different from that reported in previous studies.The mutations in BnFAD3 in the high-ALA accessions are reported for thefirst time.In identifying the sites of these mutations,we provide detailed information to aid the design of mole-cular markers for accelerated breeding schemes.

Auto-selection order of Markov chain for background sequences with chi-square test

Modeling non coding background sequences appropriately is important for the detection of regulatory elements from DNA sequences. Based on the chi square statistic test, some explanations about why to choose higher order Markov chain model and how to automatically select the proper order are given in this paper. The chi square test is first run on synthetic data sets to show that it can efficiently find the proper order of Markov chain. Using chi square test, distinct higher order context dependences inherent in ten sets of sequences of yeast S.cerevisiae from other literature have been found. So the Markov chain with higher order would be more suitable for modeling the non coding background sequences than an independent model.

Molecular characterization of LMW-GS genes from a somatic hybrid introgression line Ⅱ-12 between Triticum aestivum and Agropyron elongatum in relation to quick evolution

In order to exploit the evolution and find novel low-molecular-weight glutenin subunit （LMW-GS） for improvement of common wheat quality, thirteen variants from a somatic hybrid introgression line II-12 between Triticum aestivum cv. Jinan 177 （JN177） and Agropyron elongatum were characterized via genomic PCR. Four clones were pseudogenes because they contained an internal stop codon. The remaining nine variants contained intact open reading frames （ORFs）. Sequence alignment indicates that the proteins deduced from the nine ORFs have similar primary structure with LMW-GS cloned from its parents previously. However, they have some unique modifications in the structures. For example, EU292737 contains not only an extra Cys residue in the C-terminal domain but also a long repetitive domain. Both EU 159511 and EU292738 start their first Cys residue in the N-terminal repetitive domain, but not in the N-conserved domain traditionally. These structural alterations may have positive contributions to wheat flour quality. The results of phylogeny showed that most LMW-GS variances from 11-12 were homologous to those from parent JN177 and other wheat lines. The reason for quick evolution of LMW-GS in 11-12 was discussed.

Performance improvement of software component with partial evaluation

To avoid the complexity and inefficiency for specific applications of the current software architecture, a novel approach using partial evaluation is proposed to improve the running performance of components. The generic program was specialized into domain-specific realization for the known knowledge and environments. The syntax and semantic(adj.) were analyzed based on byte code instruction sequences, and partial evaluation rules depicted how to perform the specialization. The partial evaluation for object-oriented programs was implemented. The experimental results show that partial evaluation is effective to speed up the running efficiency. The more generality and scalability can be obtained by the integration of partial evaluation with the favorable design mechanisms and compiler optimization technology.

A CLASS OF BAND-LIMITED CHIP WAVEFORMS FOR DS-CDMA SYSTEMS

In Direct Sequence Code Division Multiple Access (DS-CDMA) systems,the chip wave-form affects the implementation,system bandwidth,envelope uniformity,eye pattern and Multiple user Access Interference (MAI). In this paper,based on an elementary density function of a second order polynomial,a class of second order continuity pulses is proposed. From this class of pulses,we can find some members having faster decaying rate,bigger eye opening,more uniform envelope and stronger anti-MAI capability than the Nyquist waveform. The normalized-bandwidth-pulse-shape-factor product,the decaying rate of the tail of the time waveform,the opening of the eye diagram,and the envelope uniformity of the second order continuity pulses are addressed in the paper that provide the basic information for the selection of the chip pulse for CDMA systems.

STUDY ON EFFICIENT COMPUTATION AND PERFORMANCE OF AV-BASED REDUCED-RANK FILTERING

In this paper, the complexity and performance of the Auxiliary Vector (AV) based reduced-rank filtering are addressed. The AV filters presented in the previous papers have the general form of the sum of the signature vector of the desired signal and a set of weighted AVs,which can be classified as three categories according to the orthogonality of their AVs and the optimality of the weight coefficients of the AVs. The AV filter with orthogonal AVs and optimal weight coefficients has the best performance, but requires considerable computational complexity and suffers from the numerical unstable operation. In order to reduce its computational load while keeping the superior performance, several low complexity algorithms are proposed to efficiently calculate the AVs and their weight coefficients. The diagonal loading technique is also introduced to solve the numerical unstability problem without complexity increase. The performance of the three types of AV filters is also compared through their application to Direct Sequence Code Division Multiple Access (DS-CDM A) systems for interference suppression.

Error correction coding paves an avenue to ultra-high accuracyof DNA sequencing

With the support from the National Natural Science Foundation of China,Prof.Huang Yanyi(黄岩谊)led a team at Peking University to demonstrate a novel approach,which combined fluorogenic sequencingby-synthesis(SBS)chemistry with an information theory-based error-correction coding scheme to

多速率DS/CDMA信号扩频序列并行盲估计方法

针对可变扩频长度多速率直接序列码分多址(direct sequence/code division multiple access,DS/CDMA)信号扩频序列盲估计问题,提出了一种基于盲源分离的并行估计方法,该方法首先利用二次功率谱的方法估计出信号不同速率的扩频码周期,然后利用多速率DS/CDMA信号周期特性将信号依据周期并行地建模为主用户和干扰用户(即将同一周期下的用户视为主用户,其余的用户和噪声一律视为独立的单个干扰用户)的模型,最后针对同步和异步两种情况,分别以单倍周期和两倍周期长度对数据进行分割,生成数据矩阵,并结合稳健的矩阵联合近似对角化(JADE)算法对扩频序列进行并行的盲估计.计算机仿真结果表明方法在较低信噪比下有效.

The Determination on Weight Hierarchies of q-Ary Linear Codes of Dimension 5 in Class IV

The weight hierarchy of a linear[n;k;q]code C over GF(q) is the sequence(d_1,d_2,…,d_k)where d_r is the smallest support of any r-dimensional subcode of C. "Determining all possible weight hierarchies of general linear codes" is a basic theoretical issue and has important scientific significance in communication system.However,it is impossible for g-ary linear codes of dimension k when q and k are slightly larger,then a reasonable formulation of the problem is modified as: "Determine almost all weight hierarchies of general g-ary linear codes of dimension k".In this paper,based on the finite projective geometry method,the authors study g-ary linear codes of dimension 5 in class IV,and find new necessary conditions of their weight hierarchies,and classify their weight hierarchies into6 subclasses.The authors also develop and improve the method of the subspace set,thus determine almost all weight hierarchies of 5-dimensional linear codes in class IV.It opens the way to determine the weight hierarchies of the rest two of 5-dimensional codes(classes III and VI),and break through the difficulties.Furthermore,the new necessary conditions show that original necessary conditions of the weight hierarchies of k-dimensional codes were not enough(not most tight nor best),so,it is important to excogitate further new necessary conditions for attacking and solving the fc-dimensional problem.